Peptide and Protein Engineering: From Concepts to Biotechnological Applications [1st ed.] 9781071607190, 9781071607206

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Olga Iranzo Ana Cecília Roque Editors

Peptide and Protein Engineering From Concepts to Biotechnological Applications

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Springer Protocols Handbooks collects a diverse range of step-by-step laboratory methods and protocols from across the life and biomedical sciences. Each protocol is provided in the Springer Protocol format: readily-reproducible in a step-by-step fashion. Each protocol opens with an introductory overview, a list of the materials and reagents needed to complete the experiment, and is followed by a detailed procedure supported by a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. With a focus on large comprehensive protocol collections and an international authorship, Springer Protocols Handbooks are a valuable addition to the laboratory.

Peptide and Protein Engineering From Concepts to Biotechnological Applications

Edited by

Olga Iranzo iSm2 UMR CNRS 7313, Aix-Marseille Université, Marseille, France

Ana Cecília Roque UCIBIO, Chemistry Department, School of Science and Technology, NOVA University of Lisbon, Caparica, Portugal

Editors Olga Iranzo iSm2 UMR CNRS 7313 Aix-Marseille Universite´ Marseille, France

Ana Cecı´lia Roque UCIBIO, Chemistry Department School of Science and Technology NOVA University of Lisbon Caparica, Portugal

ISSN 1949-2448 ISSN 1949-2456 (electronic) Springer Protocols Handbooks ISBN 978-1-0716-0719-0 ISBN 978-1-0716-0720-6 (eBook) https://doi.org/10.1007/978-1-0716-0720-6 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Illustration Caption: Image prepared by Dr. Arme´nio Barbosa. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Peptides and proteins are one of the major families of biomolecules in life. Most cellular events are regulated by protein-mediated interactions and reactions, turning the design and engineering of peptides and proteins into an important tool to control and mediate cellular functions, with important applications in the biomedical field. Furthermore, engineered peptides and proteins are also relevant for technical applications, one of the best examples being the use of enzymes and antibodies for in vitro diagnostic devices or the use of enzymes in the manufacturing of chemicals or as additives in food and cleaning agents. The wide implementation of peptides and proteins in our life has only been possible due to advances in their production following chemical routes or recombinant DNA technologies, as well as advances in molecular engineering tools to improve (or create) structure and function. Peptide and protein engineering is indeed a great scientific challenge that indeed entails a fascinating interplay among a variety of economic, social, governance, and regulatory institutions. The aim of this book from the series of Springer Protocols Handbooks is to present the methods that enabled the success of peptides and proteins in a wide variety of applications, which are covered at the biannual workshop PepperSchool. Peptide and Protein Engineering: From Concepts to Biotechnological Applications is divided in two sections. The first section comprises a collection of chapters that deal with chemical tools applied to the production or engineering of peptides and proteins. It starts with the contribution of Agouridas, Melnyk et al. describing how the bis(2-sulfanylethyl)amido (SEA) ligation, a chemoselective peptide bond-forming reaction complementary to the native chemical ligation, can be optimized at mildly acidic pH allowing a more efficient protein total synthesis. Subsequently and using the chemical synthesis of SUMO-2 and 3 proteins as examples, Melnyk et al. nicely describe how to minimize aspartimide formation during SEA ligation. In the next chapter, Agouridas et al. introduce the Protein Chemical Synthesis DataBase (http://pcs-db.fr), an accessible interactive tool with a user-friendly interface that collects the information about the chemical synthesis of proteins, using chemoselective amide-bondforming reactions, since 1994. The book continues with two chapters focused on methods using azide–alkyne cycloadditions to selectively modify biopolymers and proteins. Martins, Gomes et al. describe the immobilization of arginine-rich peptides onto aminefunctionalized chitosan using the copper(I)-catalyzed azide–alkyne cycloaddition, while Boutureira et al. report the synthesis of fluoroglycoproteins employing a metal-free protocol, i.e., a strain-promoted azide–alkyne cycloaddition. The last two chapters in this section cover protocols describing the production of peptide-based multivalent materials. Subra et al. report the synthesis of hybrid silylated peptides, both in solution and on solid support, and their subsequent use in the preparation of hybrid materials, either by a bottom-up strategy or by straightforward grafting of these hybrid silylated peptides on the surface of different materials. Finally, Pulido et al. present their approach to obtain peptideoligoethylene glycol conjugates containing different functional groups and their use to create multivalent nanomaterials, either by means of dendritic platforms or by monovalent conjugates that act as building blocks of other supramolecular structures. The second section focuses on biological approaches used to engineer structure and function in peptides and proteins. It begins with a report from Urvoas, Minard, and

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Soumillion that presents a wide variety of proteins that can be engineered by phage display, further describing methods for generating and panning highly diverse phage libraries. Be´har et al. then focus on an alternative method—ribosome display—describing in particular a method to perform ribosome display selections against targets at the surface of live bacterial cells. Dias finally shows how CIS display can be applied for the discovery of biological therapeutics detailing an improved protocol to streamline selections using this display method, thus accelerating the therapeutic discovery of novel biological drugs. Dorrazehi et al. present two methods for building gene libraries in the chromosome of Gram-negative and Gram-positive bacteria yielding chromosomal gene libraries with high diversity. Three more chapters are finally presented, which deal with the design and production of protein variants for distinct applications. Klehr et al. introduce the concept of artificial cofactors, in particular those employed to develop streptavidin-based artificial metalloenzymes. Delivoria and Skretas report a high-throughput system for identifying macrocyclic rescuers of protein misfolding. Genetically engineered bacterial cells can simultaneously perform the production of combinatorial libraries of cyclic oligopeptides and the identification of bioactive cyclic peptides that inhibit protein aggregation. Finally, Tugel, Galindo, and Wiltschi provide a protocol for the site-specific incorporation of a noncanonical amino acid with reactive side chain, as a very powerful tool for the direct chemical modification of proteins. This volume on Peptide and Protein Engineering: From Concepts to Biotechnological Applications was only possible due to the contributions from all authors. It attempts to cover the emerging principles and methodologies in peptide and protein engineering, and it is our hope that the current volume shall be of use to scientists in academia and in industry, working in areas as diverse as medicine, biology, biotechnology, diagnostics and therapeutics, and biocatalysis. Marseille, France Caparica, Portugal

Olga Iranzo Ana Cecı´lia Roque

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 SEA-Mediated Ligation Is Accelerated at Mildly Acidic pH: Application to the Formation of Difficult Peptide Junctions . . . . . . . . . . . . . . . . . . Marine Cargoe¨t, Vincent Diemer, Laurent Raibaut, Elizabeth Lissy, Benoıˆt Snella, Vangelis Agouridas, and Oleg Melnyk 2 The Problem of Aspartimide Formation During Protein Chemical Synthesis Using SEA-Mediated Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . Jennifer Bouchenna, Magalie Se´ne´chal, Herve´ Drobecq, Je´roˆme Vicogne, and Oleg Melnyk 3 Using the Interactive Tool of the Protein Chemical Synthesis Database. . . . . . . . Vangelis Agouridas and Oleg Melnyk 4 Only a “Click” Away: Development of Arginine-Rich Peptide-Based Materials Using Click Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mariana Barbosa, Fabı´ola Costa, Ca´tia Teixeira, M. Cristina L. Martins, and Paula Gomes 5 Fluoroglycoproteins by Copper-Free Strain-Promoted Azide–Alkyne Cycloaddition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pedro M. S. D. Cal, Gonc¸alo J. L. Bernardes, and Omar Boutureira 6 Hybrid Silylated Peptides for the Design of Bio-functionalized Materials . . . . . . Titouan Montheil, Ce´cile Echalier, Jean Martinez, Ahmad Mehdi, and Gilles Subra 7 Synthesis of Peptide-Oligoethylene Glycol (OEG) Conjugates for Multivalent Modification of Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel Pulido and Miriam Royo 8 Phage Display Methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agathe Urvoas, Philippe Minard, and Patrice Soumillion 9 Whole-Bacterium Ribosome Display Selection for Isolation of Antibacterial Affitins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ghislaine Be´har, Stanimir Kambarev, Jennifer Jazat, Barbara Mouratou, and Fre´de´ric Pecorari 10 CIS Display: DNA-Based Technology as a Platform for Discovery of Therapeutic Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ana Margarida Gonc¸alves Carvalho Dias 11 Building Scarless Gene Libraries in the Chromosome of Bacteria . . . . . . . . . . . . . Gol Mohammad Dorrazehi, Sebastian Worms, Jason Baby Chirakadavil, Johann Mignolet, Pascal Hols, and Patrice Soumillion

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1

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37

53 69

93 125

153

173 189

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13

14

Contents

Streptavidin (Sav)-Based Artificial Metalloenzymes: Cofactor Design Considerations and Large-Scale Expression of Host Protein Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Juliane Klehr, Jingming Zhao, Amanda Santos Kron, Thomas R. Ward, and Valentin Ko¨hler Integrated Bacterial Production and Functional Screening of Expanded Cyclic Peptide Libraries for Identifying Chemical Rescuers of Pathogenic Protein Misfolding and Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . 237 Dafni C. Delivoria and Georgios Skretas Site-Specific Incorporation of Non-canonical Amino Acids by Amber Stop Codon Suppression in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . 267 Uchralbayar Tugel, Meritxell Galindo Casas, and Birgit Wiltschi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors VANGELIS AGOURIDAS • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France MARIANA BARBOSA • LAQV-REQUIMTE, Departamento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias, University of Porto, Porto, Portugal; i3S, Instituto de Investigac¸a˜o e Inovac¸a˜o em Sau´de, University of Porto, Porto, Portugal; INEB, Instituto de Engenharia Biome´dica, University of Porto, Porto, Portugal; Faculdade de Engenharia, University of Porto, Porto, Portugal GHISLAINE BE´HAR • CRCINA, INSERM, CNRS, Universite´ d’Angers, Universite´ de Nantes, Nantes, France GONC¸ALO J. L. BERNARDES • Department of Chemistry, University of Cambridge, Cambridge, UK; Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal JENNIFER BOUCHENNA • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Lille, France ` nica, Universitat OMAR BOUTUREIRA • Departament de Quı´mica Analı´tica i Quı´mica Orga Rovira i Virgili, Tarragona, Spain PEDRO M. S. D. CAL • Department of Chemistry, University of Cambridge, Cambridge, UK; Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal MARINE CARGOE¨T • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France MERITXELL GALINDO CASAS • Austrian Centre of Industrial Biotechnology (acib GmbH), Graz, Austria; Graz University of Technology, Graz, Austria JASON BABY CHIRAKADAVIL • Biochemistry and Genetics of Microorganisms, Louvain Institute of Biomolecular Science and Technology, Universite´ catholique de Louvain, Louvain-laNeuve, Belgium FABI´OLA COSTA • i3S, Instituto de Investigac¸a˜o e Inovac¸a˜o em Sau´de, University of Porto, Porto, Portugal; INEB, Instituto de Engenharia Biome´dica, University of Porto, Porto, Portugal DAFNI C. DELIVORIA • Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece ANA MARGARIDA GONC¸ALVES CARVALHO DIAS • UCIBIO, Chemistry Department, NOVA School of Science and Technology, Caparica, Portugal; Isogenica, LLC, Cambridge, UK VINCENT DIEMER • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France GOL MOHAMMAD DORRAZEHI • Biochemistry and Genetics of Microorganisms, Louvain Institute of Biomolecular Science and Technology, Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium HERVE´ DROBECQ • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019UMR 9017-CIIL-Center for Infection and Immunity of Lille, Lille, France CE´CILE ECHALIER • IBMM Univ. Montpellier, CNRS, ENSCM, Montpellier, France; ICGM Univ. Montpellier, CNRS, ENSCM, Montpellier, France PAULA GOMES • LAQV-REQUIMTE, Departamento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias, University of Porto, Porto, Portugal

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Contributors

PASCAL HOLS • Biochemistry and Genetics of Microorganisms, Louvain Institute of Biomolecular Science and Technology, Universite´ catholique de Louvain, Louvain-laNeuve, Belgium JENNIFER JAZAT • CRCINA, INSERM, CNRS, Universite´ d’Angers, Universite´ de Nantes, Nantes, France STANIMIR KAMBAREV • CRCINA, INSERM, CNRS, Universite´ d’Angers, Universite´ de Nantes, Nantes, France JULIANE KLEHR • Department of Chemistry, University of Basel, Basel, Switzerland VALENTIN KO¨HLER • Department of Chemistry, University of Basel, Basel, Switzerland AMANDA SANTOS KRON • Department of Chemistry, University of Basel, Basel, Switzerland ELIZABETH LISSY • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France JEAN MARTINEZ • IBMM Univ. Montpellier, CNRS, ENSCM, Montpellier, France M. CRISTINA L. MARTINS • i3S, Instituto de Investigac¸a˜o e Inovac¸a˜o em Sau´de, University of Porto, Porto, Portugal; INEB, Instituto de Engenharia Biome´dica, University of Porto, Porto, Portugal; Instituto de Cieˆncias Biome´dicas Abel Salazar, University of Porto, Porto, Portugal AHMAD MEHDI • ICGM Univ. Montpellier, CNRS, ENSCM, Montpellier, France OLEG MELNYK • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France JOHANN MIGNOLET • Biochemistry and Genetics of Microorganisms, Louvain Institute of Biomolecular Science and Technology, Universite´ catholique de Louvain, Louvain-laNeuve, Belgium PHILIPPE MINARD • Protein Engineering and Modeling, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Universite´ Paris-Sud, Universite´ Paris-Saclay, Gif-surYvette, France TITOUAN MONTHEIL • IBMM Univ. Montpellier, CNRS, ENSCM, Montpellier, France BARBARA MOURATOU • CRCINA, INSERM, CNRS, Universite´ d’Angers, Universite´ de Nantes, Nantes, France FRE´DE´RIC PECORARI • CRCINA, INSERM, CNRS, Universite´ d’Angers, Universite´ de Nantes, Nantes, France DANIEL PULIDO • Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain; Institute for Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC), Barcelona, Spain LAURENT RAIBAUT • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France MIRIAM ROYO • Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain; Institute for Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC), Barcelona, Spain MAGALIE SE´NE´CHAL • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Lille, France GEORGIOS SKRETAS • Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece BENOIˆT SNELLA • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019UMR9017-CIIL-Centre d’Infection et d’Immunite´ de Lille, Lille, France PATRICE SOUMILLION • Biochemistry and Genetics of Microorganisms, Louvain Institute of Biomolecular Science and Technology, Universite´ catholique de Louvain, Louvain-laNeuve, Belgium

Contributors

xi

GILLES SUBRA • IBMM Univ. Montpellier, CNRS, ENSCM, Montpellier, France CA´TIA TEIXEIRA • LAQV-REQUIMTE, Departamento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias, University of Porto, Porto, Portugal UCHRALBAYAR TUGEL • Austrian Centre of Industrial Biotechnology (acib GmbH), Graz, Austria AGATHE URVOAS • Protein Engineering and Modeling, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Universite´ Paris-Sud, Universite´ Paris-Saclay, Gif-sur-Yvette, France JE´ROˆME VICOGNE • Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019UMR 9017-CIIL-Center for Infection and Immunity of Lille, Lille, France THOMAS R. WARD • Department of Chemistry, University of Basel, Basel, Switzerland BIRGIT WILTSCHI • Austrian Centre of Industrial Biotechnology (acib GmbH), Graz, Austria SEBASTIAN WORMS • Biochemistry and Genetics of Microorganisms, Louvain Institute of Biomolecular Science and Technology, Universite´ catholique de Louvain, Louvain-laNeuve, Belgium JINGMING ZHAO • Department of Chemistry, University of Basel, Basel, Switzerland

Chapter 1 SEA-Mediated Ligation Is Accelerated at Mildly Acidic pH: Application to the Formation of Difficult Peptide Junctions Marine Cargoe¨t, Vincent Diemer, Laurent Raibaut, Elizabeth Lissy, Benoıˆt Snella, Vangelis Agouridas, and Oleg Melnyk Abstract The bis(2-sulfanylethyl)amido (SEA)-mediated ligation has been introduced in 2010 as a novel chemoselective peptide bond-forming reaction. SEA-mediated ligation is a useful reaction for protein total synthesis that is complementary to the native chemical ligation (NCL). In particular, SEA-mediated ligation proceeds efficiently in a wide range of pH, from neutral pH to pH 3–4. Thus, the pH can be chosen to optimize the solubility of the peptide segments or final product. It can be also chosen to facilitate the formation of difficult junctions, since the rate of SEA-mediated ligation increases significantly by decreasing the pH from 7.2 to 4.0. Here we describe a protocol for SEA-mediated ligation at pH 5.5 in the presence of 4-mercaptophenylacetic acid (MPAA) or at pH 4.0 in the presence of a newly developed diselenol catalyst. The protocols describe the formation of a valyl-cysteinyl peptide bond between two model peptides. Key words bis(2-sulfanylethyl)amido, SEA-mediated ligation, Selenol catalysts, Difficult junction, pH

1

Introduction Chemical synthesis can give access to complex proteins that biological systems can hardly produce or cannot produce at all. The capacity of modern chemical methods to access challenging protein targets can be illustrated by the recent synthesis of a chaperone protein [1], of catalytically active enzymes [2, 3], or of functional ubiquitinated [4] or sumoylated [5, 6] peptide-protein conjugates. The bis(2-sulfanylethyl)amido (SEA)-mediated ligation is a useful reaction for protein total synthesis in solution [7] or on a water-compatible solid support [8, 9] that is complementary to the native chemical ligation (NCL [10–12]). The SEA-mediated ligation consists in reacting a C-terminal bis(2-sulfanylethyl)amido peptide with an N-terminal cysteinyl (Cys) peptide to produce a peptide featuring a native peptide bond to Cys (Fig. 1a) [13]. This reaction proceeds in the presence of additives such as

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Marine Cargoe¨t et al.

Fig. 1 Principle of SEA ligation (a) and the effect of the pH on the rate of SEA ligation (b)

4-mercaptophenylacetic acid (MPAA [14]) and tris(2-carboxyethyl)phosphine (TCEP). The arylthiol MPAA is used to catalyze the reaction probably through the formation of a transient arylthioester intermediate, while TCEP activates the SEA group by reduction of the cyclic disulfide and ensures that the Cys thiols are in a reduced state. Interestingly, the presence of the two thiol limbs in close proximity allows inactivating the SEA group by formation of an intramolecular disulfide bond. The SEA group in the form of its cyclic disulfide (SEAoff) is highly stable during HPLC purification, storage, or in solution in the presence of strong nucleophiles such as piperidine

Formation of Difficult Peptide Junctions by SEA Ligation

3

Fig. 2 Catalysis of SEA-mediated ligation at pH 4.0 by diselenide catalysts 5a–b

[15], aqueous bases [8], or weakly reducing thiols such as MPAA at neutral pH [16]. Due to the latter property, the SEAoff group can be used as a latent thioester during the NCL reaction provided that strong reducing agents such as TCEP or dithiothreitol (DTT) are not included in the reaction mixture [5, 8, 16–19]. The SEA group is one of the first N,S-acyl shift systems or thioester surrogates that have been shown to react with Cys peptides at neutral pH [13]. The mechanism of SEA ligation is not fully understood, and the underlying factors for this unusual reactivity remain to be established. Interestingly, the rate of SEA-mediated ligation increases significantly by decreasing the pH from 7.2 to 5.5 (Fig. 1b, see Note 1). This property can be exploited for forging difficult junctions such as the valyl-cysteinyl bond found in peptide 4b (Fig. 1a, X ¼ Val). The formation of such junctions by SEA-mediated ligation can be accomplished in two ways that are described in this protocol: by SEA-mediated ligation at pH 5.5 in the presence of MPAA (Protocol 1, Fig. 1a, see Notes 2 and 3) or by SEA-mediated ligation at pH 4.0 in the presence of diselenide 5 (Protocol 2, Fig. 2a) [20]. Figure 2b shows that the reaction in the latter

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Marine Cargoe¨t et al.

conditions proceeds faster than for the MPAA-catalyzed ligation at pH 5.5. Mechanistic studies have demonstrated that diselenide-based catalysts of type 5a–b, which are reduced in situ by TCEP into diselenols 6a–b and that are the active catalyst species, promote thiol-thioester exchanges which are rate-limiting at the working pH of 4.0 [20].

2

Materials All organic solvents and chemicals used in this protocol should be handled inside a chemical fume hood with appropriate personal protective equipment (lab coat, gloves, and protective glasses). Trifluoroacetic acid (TFA) is strongly corrosive and toxic.

2.1

General

2.2 HPLC Analysis and Purification

1. Deionized water. 1. Analytical HPLC system (Waters Alliance 2695, UV 2996 Detector, 215 nm). 2. Semi-preparative HPLC system (Waters 600 controller, UV 2487 Detector, 215 nm, TL 105 HPLC column heater). 3. Analytical HPLC column: Waters XBridge BEH300 C18 reverse-phase column (4.6  150 mm; pore size 300 A˚; particle size: 3.5 μm). 4. Semi-preparative HPLC column: Waters XBridge BEH300 C18 reverse-phase column (10  250 mm; pore size 300 A˚; particle size: 5 μm). 5. Acetonitrile for No. 412392000).

HPLC

(ACN,

Carlo

Erba,

Cat.

6. Trifluoroacetic acid (TFA; Biosolve, Cat. No. 20233301). 7. TFA 10% (vol/vol) in water. Add 10 mL of TFA carefully to 90 mL of deionized water. 8. Eluent A for semi-preparative HPLC. TFA 0.1% (vol/vol) in deionized water. Add 10 mL of TFA 10% (vol/vol) in water to 990 mL of deionized water. 9. Eluent B for semi-preparative HPLC. ACN 80% in water (vol/vol) containing 0.1% (vol/vol) TFA. Dilute 10 mL of 10% aqueous TFA (vol/vol) in 190 mL of deionized water and add 800 mL of ACN. 2.3

Lyophilization

1. Lyophilizer (Christ Gamma 2–20) equipped with a manifold. 2. Lyophilizer flasks (VWR, 300 mL, Cat. No. 88516; 600 mL, Cat. No. 88517). 3. Liquid nitrogen. 4. Dewar.

Formation of Difficult Peptide Junctions by SEA Ligation

2.4 MALDI-TOF Analysis

5

1. α-Cyano-4-hydroxycinnamic acid (Aldrich, Cat. No. 476870). 2. MALDI-TOF mass spectrometer (Autoflex Speed, Bruker). 3. A solution of α-cyano-4-hydroxycinnamic acid 10 mg/mL in 50% aqueous ACN containing 0.1% TFA.

2.5 SEA-Mediated Ligation

1. 4-Mercaptophenylacetic acid (MPAA; Alfa Aesar, Cat. No. H27658). 2. Disodium hydrogen phosphate dodecahydrate PO4∙12H2O, Merck, Cat. No. 6576).

(Na2H-

3. TFA 10% (vol/vol) in water. Add 10 mL of TFA carefully to 90 mL of deionized water. 4. Nitrogen gas (O2 < 3 ppm, Air Liquide). 5. Sodium dihydrogen phosphate dihydrate (NaH2PO4∙2H2O, Prolabo, Cat. No. 28015294). 6. Sodium hydroxide (NaOH; Merck, Cat. No. 1.06498.1000). 7. Hydrochloric acid (HCl; Sigma Aldrich, Cat. No. 32-0331500 mL). 8. Guanidine hydrochloride (Gn∙HCl; Aldrich, Cat. No. G4505). 9. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl; Aldrich, Cat. No. C4706-10G). 10. Conical tubes (15, 50 mL). 11. Glove box (Jacomex) equipped with (see Note 4): a magnetic stirrer, a block heater (37  C), a set of adjustable pipettes (0.5–10, 2–20, 10–50, 10–100, 100–1000 μL, Eppendorf), a pH meter (Eutech Instruments), and a vortex shaker. 12. Magnetic stir bars. 13. Microfuge tubes (1.5-mL safe-lock tubes; Eppendorf). 14. Pipette tips, 0.5–10, 2–20, 20–200, and 100–1000 μL (VWR). 15. 0.2 M sodium phosphate buffer pH 7.2. Prepare 36 mL of a 0.2 M solution of disodium hydrogen phosphate dodecahydrate in deionized water. Prepare 10 mL of a 0.2 M solution of sodium dihydrogen phosphate dihydrate in deionized water. Mix the two solutions and verify the pH. 16. pH paper (Merck Millipore, universal indicator 1–14, Cat. No 1.10962.0003). 17. Glacial acetic acid (Carlo Erba, 1 L, Cat. No. 401422). 18. Diethyl ether (Sigma Aldrich, Cat. No 32203-2.5L).

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Methods Peptide amides and SEAoff peptides can be prepared according to the protocols described in ref. 5.

3.1 Protocol 1: SEA-Mediated Ligation at pH 5.5 Catalyzed by MPAA

1. Weigh 4.56 mg (1 eq., 3.15 μmol) of SEAoff peptide 1b in a 1.5-mL microfuge tube. 2. Weigh 6.83 mg (1.5 eq., 4.72 μmol) of cysteinyl peptide 2 in a 1.5-mL microfuge tube. Add a magnetic stir bar to the microfuge tube. 3. Weigh 33.64 mg (0.2000 mmol) of MPAA in a 1.5-mL microfuge tube. 4. Weigh 57.33 mg (0.200 mmol) of TCEP in a 1.5-mL microfuge tube. 5. Prepare 2 mL of 0.1 M sodium phosphate buffer (pH 7.2) by diluting 1 mL of 0.2 M sodium phosphate (pH 7.2) with 1 mL of deionized water in a 15-mL plastic tube. 6. Transfer 1 mL of TFA 10% (vol/vol), 1 mL of HCl 1 M, and 1 mL of NaOH 6 M in three different 1.5-mL microfuge tubes. 7. Cap all the tubes and transfer them to the glove box under nitrogen atmosphere (see Note 5). 8. Add 1 mL of the sodium phosphate buffer from step 5 to the microfuge tube containing TCEP (see step 4). Vortex until dissolution of the TCEP, and then transfer this solution to the microfuge tube containing MPAA (see step 3). Add NaOH 6 M with the micropipette and vortex until dissolution of the MPAA (see Note 6). The final reagent concentration is 181 mM for MPAA and 181 mM for TCEP. 9. Add 450 μL of the solution prepared in step 8 to the microfuge tube containing the SEAoff peptide 1b (see step 1). Vortex to dissolve the peptide, and then add this solution to the microfuge tube containing the cysteinyl peptide 2 (see step 2). Vortex to dissolve the peptide. 10. Measure the pH of the solution prepared in step 9, and adjust to pH 5.5 (see Note 7) by adding NaOH 6 M or HCl 1 M with the micropipette (2–20 μL) (see Note 8). The final peptide concentration is ~6.7 mM for the SEAoff peptide 1b and 10 mM for the cysteinyl peptide 2. 11. Place the reaction tube from step 10 in a heating block kept at 37  C and stir with a magnetic stirrer. 12. To monitor the ligation reaction (just after preparing the reaction mixture at step 10 and every hour until 8 h of reaction and after 24, 48, 72 h, and after 7 days, see Note 9), transfer 10 μL of the reaction mixture to a microfuge tube, and quench the

Formation of Difficult Peptide Junctions by SEA Ligation

7

reaction by acidifying the sample with 20 μL of TFA 10% (vol/vol) in deionized water. Vortex and remove the sample from the glove box. 13. Remove MPAA present in the reaction mixture by extracting the aqueous phase with 1 mL of diethyl ether. Remove the diethyl ether phase with a Pasteur pipette (see Note 10). 14. Repeat step 13 twice. 15. Inject 15 μL of the aqueous sample isolated in step 14 into the analytical HPLC system to confirm that the starting peptide segments have been consumed to produce the ligation product 4b (see Note 11). 16. After 7 days, remove the reaction tube from the glove box. 17. Transfer the contents of the reaction tube into a 5-mL microfuge tube. 18. Add 550 μL of deionized water to the reaction mixture containing the crude peptide 4b and 75 μL of glacial acetic acid to a final concentration of 5–10% vol/vol with a micropipette to acidify the aqueous solution down to a pH of 2–3 (pH paper). 19. Extract the MPAA from the aqueous phase with 3 mL of diethyl ether as done in step 13. Remove the diethyl ether phase with a Pasteur pipette (see Note 10). 20. Repeat step 19 twice. 21. Remove the dissolved diethyl ether by nitrogen bubbling (~5 min). 22. Purify the target peptide 4b on a Waters XBridge BEH300 C18 reverse-phase column (10  250 mm; pore size 300 A˚; particle size: 5 μm) using a semi-preparative HPLC system; gradient: 0% eluent B in eluent A to 20% (vol/vol) eluent B in eluent A over 10 min, and then 20% (vol/vol) eluent B in eluent A to 40% (vol/vol) eluent B in eluent A over 50 min (UV detection at 215 nm). Analyze the fractions using the MALDI-TOF mass spectrometer. For this, mix 1 μL of the α-cyano-4-hydroxycinnamic acid solution with 1 μL of the sample on the MALDI plate, and let dry at room temperature in air. 23. Pool the pure fractions in a 50-mL plastic tube, freeze the solution with liquid nitrogen, and lyophilize it for 2 days. The reaction yielded 3.55 mg (55% by taking into account the samples used for analytical HPLCs) of peptide 4b. MALDI-TOF analysis: [M + H]+ calcd. 2066.2 (monoisotopic), found 2066.2.

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3.2 Protocol 2: SEA-Mediated Ligation at pH 4.0 Catalyzed by Diselenide 7b

1. Weigh 5.06 mg (1 eq., 3.49 μmol) of SEAoff peptide 1b in a 1.5-mL microfuge tube. 2. Weigh 7.53 mg (1.5 eq., 5.23 μmol) of cysteinyl peptide 2 in a 1.5-mL microfuge tube. Add a magnetic stir bar to the microfuge tube. 3. Weigh 20.76 mg of catalyst 5b (49.9 μmol) in a 1.5-mL microfuge tube (see Note 12). 4. Weigh 28.67 mg (0.1 mmol) of TCEP in a 1.5-mL microfuge tube. 5. Weigh 286.59 mg of Gn·HCl (1.200 mmol) in a 1.5-mL microfuge tube (see Note 13). 6. Prepare 1 mL of 0.1-M sodium phosphate buffer (pH 7.2) by diluting 0.5 mL of 0.2-M sodium phosphate (pH 7.2) with 0.5 mL of deionized water in a 1.5-mL microfuge tube. 7. Transfer 1 mL of TFA 0.1% in deionized water (vol/vol), 1 mL of HCl 1 M, and 1 mL of NaOH 6 M in three different 1.5-mL microfuge tubes. 8. Cap all the tubes and transfer them to the glove box under nitrogen atmosphere (see Note 5). 9. Add 300 μL of the sodium phosphate buffer from step 6 to the microfuge tube containing Gn·HCl (see step 5). Vortex until dissolution of the guanidinium salt, and transfer this solution to the microfuge tube containing TCEP (see step 4). Vortex again until dissolution of TCEP and transfer the solution to the microfuge tube containing catalyst 5b (see step 3). 10. The pH of the solution is raised to 4.0 by addition of a NaOH 6 M aqueous solution (34 μL). The final reagent concentration is 187 mM for TCEP and 94 mM for catalyst 5b. 11. Add 499 μL of the solution prepared in step 10 to the microfuge tube containing the SEAoff peptide 1b (see step 1). Vortex to dissolve the peptide and then transfer this solution to the microfuge tube containing the cysteinyl peptide 2 (see step 2). Vortex to dissolve the peptide. 12. Place the reaction tube in a heating block kept at 37  C and stir with a magnetic stirrer (see Note 14). 13. To monitor the ligation reaction (just after preparing the reaction mixture at step 11 and at regular time intervals until completion of the reaction), transfer 1 μL of the reaction mixture to a microfuge tube containing 50 μL of TFA 0.1% (vol/vol) in deionized water. Vortex and remove the sample from the glove box (see Note 15). 14. Inject 30 μL of the reaction sample isolated in step 13 into the analytical HPLC system to confirm that the starting peptide segments have been consumed to produce the ligation product 4b.

Formation of Difficult Peptide Junctions by SEA Ligation

9

15. After completion of the reaction, remove the reaction tube from the glove box. 16. Transfer the contents of the reaction tube into a 5-mL microfuge tube, and add 5 mL of a 0.1% TFA solution in deionized water (v/v). Filter the crude solution through a disposable syringe filter before injection. 17. Purify the target peptide 4b on a Waters XBridge BEH300 C18 reverse-phase column (10  250 mm; pore size 300 A˚; particle size: 5 μm) using a semi-preparative HPLC system; gradient: 0% eluent B in eluent A to 20% (vol/vol) eluent B in eluent A over 5 min, and then 20% (vol/vol) eluent B in eluent A to 35% (vol/vol) eluent B in eluent A over 60 min (UV detection at 215 nm). Analyze the fractions using the MALDI-TOF mass spectrometer. For this, mix 1 μL of the α-cyano-4-hydroxycinnamic acid solution with 1 μL of the sample on the MALDI plate, and let dry at room temperature in air. 18. Pool the pure fractions in a 50-mL plastic tube, freeze the solution with liquid nitrogen and lyophilize it for 2 days. The reaction yielded 4.69 mg (51%) of peptide 4b. MALDI-TOF analysis: [M + H]+ calcd. 2066.2 (monoisotopic), found 2066.5.

4

Notes 1. The SEA group can be introduced on the side chain of aspartic or glutamic acid residues for accessing branched or tail to sidechain cyclized peptides. In this case also, a significant acceleration of the SEA ligation process has been noticed by lowering the pH from 7.3 to 5.5 [15]. 2. For an application of SEA-mediated ligation at pH 5.5 to the synthesis of large cyclic peptides, see ref. 21. 3. Peptidyl prolyl thioesters, i.e., peptide1-AA-Pro-SR (R ¼ alkyl), are known to be poorly reactive toward Cys peptides (Cys-peptide2) [22]. A similar observation has been made for peptidyl prolyl SEA peptides (peptide1-AA-Pro-SEA) [23]. However, the rate of the SEA ligation process can be increased significantly by raising the temperature to 65  C and conducting the experiment at pH 5.5. Moreover, we have also shown that the target ligation product of peptidyl prolyl thioesters or peptidyl prolyl SEA peptides with Cys peptides, i.e., peptide1-AA-ProCys-peptide2, can be contaminated by substantial amounts of a two-amino-acid deletion side product, i.e., peptide1-Cyspeptide2, corresponding to the loss of AA-Pro residues. Sideproduct formation is probably due to the degradation of the

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peptidyl prolyl SEA or thioester peptide through intramolecular cyclization and formation of a transient N-peptidyl diketopiperazine intermediate. Interestingly, conducting the SEA ligation process at pH 5.5 allowed reducing significantly the proportion of the deletion side product in comparison with the peptidyl prolyl thioester analog used at pH 7.1. 4. The glove box used in this work allows performing the ligation experiments in a nitrogen atmosphere with oxygen levels below 10 ppm. Alternatively, an inflatable plastic glove bag can be used. Very low oxygen levels are not mandatory for performing SEA-mediated ligations although it is recommended when long reaction times are needed as for the synthesis of peptide 4b [22]. Molecular oxygen oxidizes thiolate species into disulfides, which are in turn reduced by TCEP. The net result of this process is that molecular oxygen consumes TCEP which allows maintaining the thiols in a reduced form. If the HPLC chromatograms of the reaction mixture show the formation of disulfides, and in particular of mixed disulfides with MPAA (see ref. 19), more TCEP must be added to the reaction mixture. 5. The tubes must be tightly capped during transfer into the glove box, which requires purging the airlock by pumping and filling with nitrogen. 6. TCEP hydrochloride is a highly acidic reagent. Dissolution of TCEP in the phosphate buffer results in a significant decrease of the pH well below pH 5.5. MPAA is poorly soluble in these conditions. MPAA is highly soluble in water at and above pH 5.5 due to the ionization of its thiol acid group (the pKa of MPAA thiol is 6.6) [14]. MPAA will therefore solubilize gradually upon addition of NaOH and vortexing. At this stage, a precise control of the pH is not mandatory. However, the addition of NaOH must be done carefully to avoid a significant increase of the pH, which would require the addition of large volumes of HCl at step 10. 7. SEA-mediated ligation well below pH 5.5 in the presence of MPAA is feasible and can be very useful. For example, SEA-mediated ligation at pH 3.0 allowed the synthesis of Oacyl isodipeptides without any protection on the O-acyl isodipeptide units because at this pH the free amino group of the Oacyl isodipeptide units is protected by protonation [24]. The solubility of MPAA at pH 3.0 in the presence of 0.6 M guanidinium hydrochloride is ~30 mM [24]. 8. The addition of NaOH 6 M (or HCl 1 M) must be done carefully; otherwise the pH can raise significantly above (or decrease well below) pH 5.5. 9. Ligation at valine is notoriously difficult (see ref. 22).

Formation of Difficult Peptide Junctions by SEA Ligation

11

10. The large excess of MPAA used for the SEA-mediated ligation must be extracted with diethyl ether before running the analytical or the semi-preparative HPLC systems. This arylthiol absorbs strongly at the wavelengths used for the UV detection by the HPLC systems and can co-elute with the target peptide. The efficient extraction of MPAA requires the acidification of the mixture well below its pKa. This can be achieved by adding TFA 10% (vol/vol) to the reaction mixture before the extraction. An alternative is to use acetic acid (5–10% vol/vol final concentration). 11. MPAA catalyzes the SEA-mediated ligation process through probably the in situ formation of an arylthioester peptide segment by a thiol-thioester exchange mechanism [25]. Usually, the arylthiolester peptide derived from the SEA peptide reacts quickly with the cysteinyl component so that it is usually not observed in the HPLC traces of the crude ligation mixture. Nevertheless, it can be observed when the SEA peptide is used in excess relative to the Cys peptide. 12. Catalysts 5a and 5b can be used without any significant difference with regard to the kinetics and final yield of the ligation reaction (see ref. 20). 13. Guanidine hydrochloride is a strong peptide denaturant. It is used classically at 6 M concentration to avoid any partial folding of the peptide segments that could mask reactive ends during ligation and affect ligation efficiency. 14. Deselenization of the catalyst during ligation is insignificant [20]. 15. The use of type 5 diselenide catalysts in the reaction considerably simplifies the workup as they are highly soluble in water. Contrary to MPAA, no extractive workup is necessary prior to HPLC injection.

Acknowledgments This study was supported by the Centre National de la Recherche Scientifique (CNRS), the University of Lille Nord de France, and the Institut Pasteur de Lille. References 1. Weinstock MT, Jacobsen MT, Kay MS (2015) Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity. Proc Natl Acad Sci U S A 111:11679–11684 2. Wintermann F, Engelbrecht S (2013) Reconstitution of the catalytic core of F-ATPase

(ab)3γ from Escherichia coli using chemically synthesized subunit γ. Angew Chem Int Ed 52:1309–1313 3. Lahiri S, Brehs M, Olschewski D, Becker CF (2011) Total chemical synthesis of an integral membrane enzyme: diacylglycerol kinase from

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Escherichia coli. Angew Chem Int Ed 50:3988–3992 4. Siman P, Karthikeyan SV, Nikolov M, Fischle W, Brik A (2013) Convergent chemical synthesis of histone H2B protein for the sitespecific ubiquitination at Lys34. Angew Chem Int Ed 52:8059–8063 5. Boll E, Drobecq H, Ollivier N, Blanpain A, Raibaut L, Desmet R, Vicogne J, Melnyk O (2015) One-pot chemical synthesis of small ubiquitin-like modifier (SUMO) proteinpeptide conjugates using bis(2-sulfanylethyl) amido peptide latent thioester surrogates. Nat Protoc 10:269–292 6. Boll E, Drobecq H, Ollivier N, Raibaut L, Desmet R, Vicogne J, Melnyk O (2014) A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1 peptide conjugate. Chem Sci 5:2017–2022 7. Raibaut L, Ollivier N, Melnyk O (2012) Sequential native peptide ligation strategies for total chemical protein synthesis. Chem Soc Rev 41:7001–7015 8. Raibaut L, Adihou H, Desmet R, Delmas AF, Aucagne V, Melnyk O (2013) Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segments. Chem Sci 4:4061–4066 9. Raibaut L, El Mahdi O, Melnyk O (2015) Solid phase protein chemical synthesis. Top Curr Chem 363:103–154 10. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779 11. Kent SBH (2009) Total chemical synthesis of proteins. Chem Soc Rev 38:338–351 12. Agouridas V, El Mahdi O, Diemer V, Cargoet M, Monbaliu J-CM, Melnyk O (2019) Native chemical ligation and extended methods. Mechanisms, catalysis, scope and limitations. Chem Rev 12:7328–7443 13. Ollivier N, Dheur J, Mhidia R, Blanpain A, Melnyk O (2010) Bis(2-sulfanylethyl)amino native peptide ligation. Org Lett 12:5238–5241 14. Johnson EC, Kent SBH (2006) Insights into the mechanism and catalysis of the native chemical ligation reaction. J Am Chem Soc 128:6640–6646

15. Boll E, Dheur J, Drobecq H, Melnyk O (2012) Access to cyclic or branched peptides using bis (2-sulfanylethyl)amido side-chain derivatives of Asp and Glu. Org Lett 14:2222–2225 16. Ollivier N, Vicogne J, Vallin A, Drobecq H, Desmet R, El-Mahdi O, Leclercq B, Goormachtigh G, Fafeur V, Melnyk O (2012) A one-pot three-segment ligation strategy for protein chemical synthesis. Angew Chem Int Ed 51:209–213 17. Raibaut L, Drobecq H, Melnyk O (2015) Selectively activatable latent thiol and selenolesters simplify the access to cyclic or branched peptide scaffolds. Org Lett 17:3636–3639 18. Melnyk O, Agouridas V (2014) Perhydro1,2,5-dithiazepine. e-EROS, No rn01723 19. Raibaut L, Vicogne J, Leclercq B, Drobecq H, Desmet R, Melnyk O (2013) Total synthesis of biotinylated N domain of human hepatocyte growth factor. Bioorg Med Chem 21:3486–3494 20. Cargoe¨t M, Diemer V, Snella B, Desmet R, Blanpain A, Drobecq H, Agouridas V, Melnyk O (2018) Catalysis of thiol-thioester exchange by water-soluble alkyldiselenols applied to the synthesis of peptide thioesters and SEA-mediated ligation. J Org Chem 83:12584–12594 21. Boll E, Ebran JP, Drobecq H, El-Mahdi O, Raibaut L, Ollivier N, Melnyk O (2015) Access to large cyclic peptides by a one-pot two-peptide segment ligation/cyclization process. Org Lett 17:130–133 22. Hackeng TM, Griffin JH, Dawson PE (1999) Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc Natl Acad Sci U S A 96:10068–10073 23. Raibaut L, Seeberger P, Melnyk O (2013) Bis (2-sulfanylethyl)amido peptides enable native chemical ligation at proline and minimize deletion side-product formation. Org Lett 15:5516–5519 24. Desmet R, Pauzuolis M, Boll E, Drobecq H, Raibaut L, Melnyk O (2015) Synthesis of unprotected linear or cyclic O-acyl isopeptides in water using bis(2-sulfanylethyl)amido peptide ligation. Org Lett 17:3354–3357 25. Dawson PE, Churchill MJ, Ghadiri MR, Kent SBH (1997) Modulation of reactivity in native chemical ligation through the use of thiol additives. J Am Chem Soc 119:4325–4329

Chapter 2 The Problem of Aspartimide Formation During Protein Chemical Synthesis Using SEA-Mediated Ligation Jennifer Bouchenna, Magalie Se´ne´chal, Herve´ Drobecq, Je´roˆme Vicogne, and Oleg Melnyk Abstract Aspartimide formation often complicates the solid-phase synthesis of peptides. Much less discussed is the potential occurrence of this side reaction during the coupling of peptide segments using chemoselective peptide bond-forming reactions such as the native chemical ligation and extended methods. Here we describe how to manage this problem using bis(2-sulfenylethyl)amido (SEA)-mediated ligation. Key words Aspartimide, bis(2-Sulfenylethyl)amido (SEA)-mediated ligation, SPPS

1

Introduction Aspartimide formation is a well-known side reaction, which often complicates the solid-phase peptide synthesis (SPPS [1]). This side reaction is due to the carboxylic acid group of the aspartic acid side chain, which can react with the α-nitrogen of the next residue to form a succinimide moiety called aspartimide. The aspartimide can eventually be opened by nucleophiles, typically water or hydroxide ion, to produce a peptide having a natural backbone or an isopeptidic structure depending on which aspartimide carbonyl is attacked by the nucleophile. Note that the opening of an aspartimide yields usually the isopeptide as the major product. Note also that aspartimides are prone to epimerization. Therefore, aspartimide formation can ultimately result in the formation of several by-products. Several tools are now available for minimizing this side reaction during SPPS such as optimized Fmoc deprotection cocktails, sidechain protecting groups for aspartic acid, or backbone protecting groups for the residue following Asp [2]. Aspartimide by-product formation can also spontaneously occur in aqueous solution at neutral pH and as such is one of the

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Fig. 1 Sequences of SUMO-2 and SUMO-3 proteins showing the presence of an Asp-Gly dipeptide unit on both sides of the ligation site

mechanisms that lead to the degradation of proteins in vivo with aging [3]. The occurrence of this side reaction in proteins [3], peptide therapeutics [4], and model peptides [5–7] featuring Asn or Asp residues has been the subject of numerous studies. The occurrence of Asp/Asn-Gly dipeptides in proteins, which are particularly prone to aspartimide formation, is on average 2.7 units per protein (1.2 for Asp-Gly, 1.5 for Asn-Gly, data extracted from UniRef50 [8]). Therefore, such aspartimide-prone sites are frequent and can considerably complicate the synthesis of most proteins (see Note 1), regardless of the type of ligation used [9]. The chemical synthesis of SUMO-2 and SUMO-3 proteins has been described in a few works using either ketoacid-hydroxylamine ligation [10] or optimized SPPS protocols [11]. In each case, the SPPS of the peptide segments was complicated by aspartimide by-product formation due to the presence of two Asp-Gly units in SUMO-2 and SUMO-3 domains (Fig. 1). Aspartimide by-product formation during SPPS could be suppressed by using Fmoc-Asp(OtBu)-(Dmb) Gly-OH dipeptide unit for introducing the Asp and Gly residues. During our investigations on the chemical synthesis of SUMO2 and SUMO-3 proteins by SEA-mediated ligation [12], we also found that the use of Fmoc-Asp(OtBu)-(Dmb)Gly-OH dipeptide unit during the SPPS of the peptide segments was mandatory for suppressing aspartimide by-product formation. Performing the ligation of the peptide segments under classical experimental conditions (pH 5.5, 4-mercaptophenylacetic acid (MPAA) [13] 37
C) resulted in the concomitant formation of the target SUMO protein and of a 18 uma contaminant (~20% by MALDI-TOF), which could not be separated by HPLC. Interestingly, lowering the temperature of the ligation mixture to 25
C enabled greatly minimizing the side reaction and the isolation of the target proteins in good yield and purity [14, 15]. This protocol details the chemical synthesis of SUMO-2 and SUMO-3 proteins by SEA-mediated ligation at 25
C in the presence of MPAA. The presence in SUMO proteins of a Cys residue in central position enabled their assembly by ligating two peptide segments of about 45 amino acids (Fig. 1). The N-terminal peptide segments, i.e., SUMO-xN (x ¼ 2 or 3), were produced by 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS using bis(2-sulfanylethyl)amino (SEA) ChemMatrix® resin (Fig. 2a) [16, 17]. The

Aspartimide Formation During SEA-Mediated Ligation

15

Fig. 2 Synthesis of SUMO-2 and SUMO-3 proteins by SEA-mediated ligation. (a) SPPS of the N-terminal segments using SEA-ChemMatrix® resin. (b) SPPS of the C-terminal segment using 4-hydroxymethylphenoxyacetyl (HMPA) ChemMatrix® resin. (c) Assembly of SUMO-2 and SUMO-3 proteins by SEA-mediated ligation at 25
C

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C-terminal peptide segment SUMO-2/3C is the same for both proteins. It was produced by Fmoc SPPS starting from 4-hydroxymethylphenoxyacetyl (HMPA) ChemMatrix® resin (Fig. 2b).

2

Materials and Instruments All organic solvents and chemicals used in this protocol should be handled inside a chemical fume hood with appropriate personal protective equipment (lab coat, gloves, and protective glasses). Trifluoroacetic acid (TFA) is strongly corrosive and toxic.

2.1

General

1. Synthesis of bis(2-sulfanylethyl)aminotrityl ChemMatrix (SEA ChemMatrix) resin was carried out as described elsewhere [16, 17]. 2. HMPA ChemMatrix resin (Iris Biotech, Lot 08K11-20-03043). 3. Dichloromethane RPE ACS stabilized with amylene (DCM; Carlo Erba, Cat. No. 463314). 4. N,N-Dimethylformamide for peptide synthesis (DMF; Carlo Erba, Cat. No. P0343521). 5. 1-Methyl-2-pyrrolidinone No. P0873521).

(NMP;

Carlo

Erba,

Cat.

6. Resins were conditioned in DCM (3  2 min, 3 mL) and then in DMF (3  2 min, 3 mL) in a manual SPPS glass reactor prior to their use. 7. Nα-Fmoc-protected amino acids were obtained from Iris Biotech GmbH: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu) [Dmb-Gly]-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, FmocPhe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Cys (StBu)-OH, or Fmoc-Cys(Trt)-OH. 8. N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1ylmethylene]-N methylmethanaminium hexafluorophosphate N-oxide (HATU; Novabiochem, Cat. No. 01-62-0041). 9. N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HBTU; Iris Biotech, Cat. No. RL-1030). 10. N,N-Diisopropylethylamine (DIEA; SDS, Cat. No. 0403516). 11. Acetaldehyde (Aldrich, Cat. No. 110078), 2 vol-% in DMF.

Aspartimide Formation During SEA-Mediated Ligation

17

12. p-Chloranil (Acros, Cat. No. 213561000), 2 wt-% in DMF. 13. 2,4,6-Trinitrobenzenesulfonic acid (TNBS,TIC Europe N.V, Cat. No. 2508-19-2). 14. N,N0 -diisopropylcarbodiimide No. 446181000).

(DIC,

Acros,

Cat.

15. 4-(Dimethylamino)pyridine (DMAP, Aldrich Chemie, Cat. No. 10 770-0). 16. Acetic anhydride (Ac2O) was purchased from BioSolve and Fisher Chemical. 17. UV spectrophotometer. 18. Precision quartz cell (length 10 mm, quartz Suprasil, Hellma, Cat. No. 117104-05). 19. Diethyl ether RPE stabilized with 2,6-bis(1,1-dimethylethyl)4-methylphenol (Carlo Erba, Cat. No. 447523). 20. Piperidine for No. 16183302).

peptide

synthesis

(BioSolve,

Cat.

21. Thioanisole (Fluka, Cat. No. 88470). 22. Thiophenol (Alfa Aesar, Cat. No.10201873) 23. Trifluoroacetic acid (TFA; BioSolve, Cat. No. 20233301). 24. Triisopropylsilane (TIS; Aldrich, Cat. No. 23378-1). 25. 1,2-Ethanedithiol, 99% (EDT, Aldrich, Cat. No. E360-0). 26. n-Heptane (BioSolve, Cat. No. 0805202). 27. Water was purified with a Milli-Q Ultrapure Water Purification System. 28. Dithiothreitol (DTT; Acros Organics, Cat. No. 16568-0050). 29. Iodine (Acros Organics, Cat. No. 38705100). 30. 0.1% TFA in water, Optima LC/MS (Fisher Chemical, Cat. No. LS119-212). 31. 0.1% TFA in acetonitrile, Optima LC/MS (Fisher Chemical, Cat. No. LS119-212). 32. Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, Prolabo, Cat. No. 28015294). 33. Disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O, Prolabo, Cat. No. 28015294). 34. Guanidine hydrochloride (Gn∙HCl; Aldrich, Cat. No. G4505). 35. Glacial acetic acid (AcOH) (Carbo Erba, Cat. No. 401422). 36. Nitrogen gas (O2 < 3 ppm, Air Liquide). 37. Argon (Alphagaz, Air Liquide). 38. Hydrochloric acid (HCl; Sigma Aldrich, Cat. No. 32-0331500 mL).

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39. Sodium hydroxide (NaOH; VWR, Cat. No. 28240292). 40. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP∙HCl; Aldrich, Cat. No. C4706-10G). 41. 4-Mercaptophenylacetic acid (MPAA; Alfa Aesar, Cat. No. H27658). 42. Conical tubes (15 mL, 50 mL). 43. Thermomixer comfort (Eppendorf) equipped with a block heater (25
C). 44. A set of adjustable pipettes (0.5–10, 2–20, 10–50, 10–100, 100–1000 μL, Eppendorf). 45. Pipette tips, 0.5–10, 2–20, 20–200, and 100–1000 μL (VWR). 46. pH meter. 47. Vortex shaker. 48. Microfuge tubes (1.5-mL safe-lock tubes; Eppendorf). 2.2 HPLC Analysis and Purification

1. TFA 10% (vol/vol) in water. Add carefully 10 mL of TFA to 90 mL of deionized water. 2. Eluent A for semi-preparative HPLC. TFA 0.1% (vol/vol) in deionized water. Add carefully 10 mL of 10% aqueous TFA (vol/vol) to 990 mL of deionized water. 3. Eluent B for semi-preparative HPLC. ACN 80% (vol/vol) containing 0.1% (vol/vol) TFA. Add 10 mL of 10% aqueous TFA (vol/vol) to 990 mL of ACN 80% (vol/vol) in deionized water. 4. Analytical UPLC-MS system: Dionex UltiMate 3000/LCQ Fleet Ion Trap, Dionex DA detector (215–280 nm), Corona Veo charged aerosol detector, ES+, m/z range 300–2000, capillary voltage 2.8 kV, cone voltage 10 V, tube lens 75 V, capillary voltage temperature 350
C. 5. Analytical UPLC-MS column: Acquity UPLC peptide BEH300 C18 reverse-phase column (2.1  100 mm; pore size 300 A˚; particle size: 1.7 μm). 6. Semi-preparative HPLC system (Waters 600 controller, UV 2487 Detector, 215 nm, TL 105 HPLC column heater). 7. Semi-preparative HPLC column: Waters XBridge BEH300 C18 reverse-phase column (10  250 mm; pore size 300 A˚; particle size: 5 μm).

2.3

Lyophilization

1. Lyophilizer (Christ Gamma 2-20) equipped with a manifold. 2. Lyophilizer flasks (VWR, 300 mL, Cat. No. 88516; 600 mL, Cat. No. 88517). 3. Liquid nitrogen. 4. Dewar.

Aspartimide Formation During SEA-Mediated Ligation

2.4 MALDI-TOF Analysis

19

1. MALDI-TOF mass spectrometer (Autoflex Speed, Bruker). 2. 2,5-Dihydroxybenzoic acid (DHB, Aldrich, Cat. No. 490799). 3. A solution of DHB 20 mg/mL in 50% aqueous ACN containing 0.1% TFA.

3

Methods

3.1 Solid-Phase Peptide Synthesis of the Peptide Segments

1. Column peptide synthesizer (MultiPep Intavis AG Bioanalytical instrument equipped with a six-column module). 2. Synthesis columns (Intavis, Cat. No. 35760). 3. Reaction columns (Intavis, Cat. No. 34274). 4. Manual peptide synthesis vessels. 5. Fmoc amino acid solutions, Fmoc-AA-OH 0.54 M in DMF. 6. DIEA solution, DIEA 35% in NMP (vol/vol). 7. HBTU solution: HBTU 0.54 M in DMF. 8. Piperidine solution 20% in DMF (vol/vol) (see Note 2). 9. Capping solution, acetic anhydride 10%/DIEA 5%/DMF 85% (vol/vol/vol) (see Note 3).

3.1.1 Coupling of First Amino Acid to the SEA-Trt ChemMatrix® Resin

1. Weigh the amino acid Fmoc-Tyr(OtBu)-OH (459 mg, 1.00 mmol, 10 equiv) in a plastic tube. 2. Weigh 377 mg (0.95 mmol, 9.5 equiv) of HATU in a plastic tube (see Note 4). 3. Add 5 mL of DMF to the tubes and agitate with a vortex shaker to dissolve the reagents. 4. Transfer the HATU/DMF solution to the tube containing the amino acid solution with an adjustable pipette. 5. Add 348 μL (2.00 mmol, 20 equiv) of DIEA to the tube with an adjustable pipette, and agitate with a vortex shaker for 1 min. The solution becomes yellow upon addition of the base. 6. Transfer the activated amino acid/DMF solution to the reaction column. 7. Agitate with a laboratory flask shaker for 1 h 30 min and drain the column. 8. Add 5–10 mL of DMF, agitate for 2 min, and then drain the column. 9. Repeat step 8 twice. 10. Take a sample of the resin and perform the chloranil test which must be negative (colorless resin beads). 11. Acetylate remaining amino groups eventually present on the Fmoc-Tyr(OtBu)-SEA-Trt ChemMatrix resin by adding 5 mL

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of acetic anhydride/DIEA/DMF capping solution to the resin. Shake for 10 min and drain (see Note 5). 12. Repeat step 11 twice. 13. Wash the resin with DMF (3  2 min, 5 mL). 14. Wash the resin with DCM (3  2 min, 5 mL). 15. Add 5 mL of diethyl ether, shake for 2 min, and then drain the column. 16. Repeat step 15 twice. 17. Dry the beads in vacuo. 18. Determine the loading of the Fmoc-Tyr(OtBu)-SEA-Trt ChemMatrix resin by treating the resin with the 20% piperidine solution in DMF and UV quantification of the formed dibenzofulvene-piperidine adduct at 290 nm as described elsewhere [16]. Typical loading for Fmoc-Tyr(OtBu)-SEA-Trt ChemMatrix resin: 0.22 mmol/g. 3.1.2 Coupling of First Amino Acid to the HMPA ChemMatrix® Resin

1. Weigh HMPA ChemMatrix® resin (500 mg). 2. Weigh the amino acid Fmoc-Gly-OH (297 mg, 1.00 mmol) in a plastic tube. 3. Weight 12.2 mg (0.100 mmol) of DMAP. 4. Add 5 mL of DMF to the tube containing the amino acid to dissolve it. 5. Add 78.3 μL (0.500 mmol) of DIC to the tube containing the amino acid solution with an adjustable pipette. 6. Transfer the activated amino acid/DIC solution to the reaction column. 7. Add DMAP in one portion to the reaction column. 8. Agitate with a laboratory flask shaker for 2 h at room temperature. 9. Add 5–10 mL of DMF, agitate for 2 min, and drain the column. 10. Repeat step 9 twice. 11. Wash the resin with DMF (3  2 min, 5 mL). 12. Wash the resin with DCM (3  2 min, 5 mL). 13. Add 5 mL of diethyl ether, shake for 2 min, and drain the column. 14. Repeat step 13 twice. 15. Determine the loading of the Fmoc-Gly-HMPA ChemMatrix® resin after treating an aliquot with 20% piperidine solution in DMF and reading the absorbance of the supernatant at 290 nm. Typical loading for Fmoc-Gly-HMPA ChemMatrix® resin: 0.58 mmol/g.

Aspartimide Formation During SEA-Mediated Ligation 3.1.3 First Elongation Using the Automated Peptide Synthesizer

21

Elongate Fmoc-Tyr(OtBu)-SEA-Trt ChemMatrix resin and FmocGly-HMPA ChemMatrix® resin using the automated peptide synthesizer (see Note 6). Elongated sequence using Fmoc-Tyr(OtBu)-SEA-Trt ChemMatrix resin (SUMO-2N and SUMO-3N): SVVQFKIKRHTPLSKLMKA. Elongated sequence using Fmoc-Gly-HMPA ChemMatrix® resin (SUMO-2/3C): QPINETDTPAQLEMEDEDTIDVFQQQTG.

3.1.4 Manual Coupling of Fmoc-Asp(OtBu)[Dmb] Gly-OH

1. Weigh Fmoc-Asp(OtBu)[Dmb]Gly-OH 0.300 mmol, 3 equiv) in a plastic tube.

(186

mg,

2. Weigh 108 mg of HATU (108 mg, 0.284 mmol, 2.85 equiv) in the plastic tube. 3. Add 3 mL of DMF to the tube containing HATU and agitate with a vortex shaker to dissolve the reagent. 4. Transfer the HATU/DMF solution to the tube containing the amino acid with an adjustable pipette. 5. Add 104 μL of DIEA (0.600 mmol, 6 equiv) to the tube with an adjustable pipette, and agitate with a vortex shaker for 1 min. The solution becomes yellow upon addition of the base. 6. Transfer the activated dipeptide unit/DMF solution to the reaction column. 7. Agitate with a laboratory flask shaker for 2 h and then drain the column. 8. Add 5–10 mL of DMF, agitate for 2 min, and drain the column. 9. Repeat step 8 twice. 10. Take a sample of the resin and perform a TNBS test that must be negative (colorless resin beads) (see Note 7). 11. Optional: analysis of the elongated peptide by MALDI-TOF after cleavage and deprotection (see steps 12–14). 12. Treat an aliquot of the peptidyl resin with 200 μL of 20% piperidine to remove the N-terminal Fmoc group. 13. Remove the piperidine solution with 1 mL of diethyl ether using a Pasteur pipette, and dry the resin under a gentle flow of argon. 14. Perform the TFA cleavage for 1 h by adding 70 μL of the cleavage mixture to the resin aliquot: TFA/triisopropylsilane (TIS)/thioanisole/H2O/thiophenol, 87.5/5/2.5/2.5/2.5 by vol (prepare 1 mL) for SEA ChemMatrix® resin, or TFA/TIS/H2O/ethanedithiol (EDT), 90/5/2.5/2.5 by vol (prepare 1 mL) for HMPA-ChemMatrix® resin.

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Take 1 μL of the cleavage mixture, dilute with water (50 μL), and analyze by MALDI-TOF to confirm the successful elongation of the peptide chain including the coupling of the dipeptide unit. Elongated sequence using Fmoc-Tyr (OtBu)-SEA-Trt ChemMatrix resin (SUMO-2N and SUMO3N): DGSVVQFKIKRHTPLSKLMKAY-SEAon [M+H]+ calc. 2663.45, found 2664.86. Elongated sequence using Fmoc-Gly-HMPA ChemMatrix® resin (SUMO-2/3C): DGQPINETDTPAQLEMEDEDTIDVFQQQTGG-OH [M +H]+ calc. 3421.48, found 3422.10. See Note 8. 15. Continue the synthesis by acetylating unreacted amino groups eventually present on the HMPA or SEA ChemMatrix® peptidyl resins by adding 5 mL of acetic anhydride/DIEA/DMF capping solution to the solid support. Shake for 10 min and drain. 16. Repeat step 15 twice. 17. Add 5 mL of DMF to the column, shake for 2 min, and drain the column. 18. Repeat step 17 twice. 3.1.5 Second Elongation Using Automated Peptide Synthesizer

Elongate the peptidyl resins using the automated peptide synthesizer. Elongated sequence for SEA-Trt ChemMatrix resin: ADEKPKEGVKTENNDHINLKVAGQ for SUMO-2N, SEEKPKEGVKTENDHINLKVAGQ for SUMO-3N. Elongated sequence for HMPA ChemMatrix® CERQGLSMRQIRFRF for SUMO-2/3C.

3.1.6 Cleavage Step

resin:

1. In a graduated cylinder, add 8.75 mL of TFA, 0.25 mL of deionized water, 0.25 mL of thiophenol, 0.25 mL of thioanisole, and 0.50 mL of TIS for peptides SUMO-xN (x ¼ 2, 3). 2. In a graduated cylinder, add 9 mL of TFA, 0.25 mL of deionized water, 0.25 mL of EDT, and 0.50 mL of TIS for peptide SUMO-2/3C. 3. Transfer each peptidyl resin in a different manual peptide synthesis vessel. 4. Add the TFA solution to the manual peptide synthesis vessel and agitate at room temperature for 2 h. 5. In the meantime, prepare three 300-mL Erlenmeyer flasks, each containing a mixture of 150 mL of diethyl ether and 150 mL of n-heptane. Add magnetic bars and place the Erlenmeyer flasks in an ice bath.

Aspartimide Formation During SEA-Mediated Ligation

23

6. Precipitate the peptides by adding the peptide solutions dropwise to the ice-cold diethyl ether/n-heptane mixture with magnetic stirring. 7. Add 5 mL of TFA to each peptide reactor, shake the bead suspensions for 2 min, and then add this solution dropwise to the Erlenmeyer flask. 8. Repeat step 7 (see Note 9). 9. Transfer the suspensions to centrifuge tubes and centrifuge at 1900  g for 10 min, then pour carefully the supernatant into an Erlenmeyer flask. 10. Add 40 mL of cold diethyl ether 50%/n-heptane 50% (vol/vol) to the peptide, triturate the peptide pellet, centrifuge again at 1900  g for 10 min, and then pour carefully the supernatant into the Erlenmeyer flask. 11. Carefully flush the tubes with nitrogen to evaporate the residual solvent. 12. Dissolve the crude peptides in TFA 0.1% (vol/vol) in deionized water, and place the peptide solutions immediately at 20
C overnight (see Note 10). 13. Lyophilize for 2 days. 14. Analyze the crude peptide by UPLC-MS and MALDI-TOF mass spectrometry. 3.1.7 Oxidation SEAon Peptides: Purification of SUMO-xN SEAoff Peptides (x ¼ 2,3)

1. Prepare the semi-preparative HPLC system, which should be ready before starting the oxidation of the peptides. 2. Rapidly weigh 100–150 mg of iodine in a 5-mL volumetric flask. Immediately add the DMSO up to 5 mL. Calculate the concentration of iodine (see Note 11). 3. Prepare a solution of DTT 10 mg/mL in deionized water. 4. Dissolve the crude peptide in phosphate buffer/Gn∙HCl 6 M at room temperature (peptide concentration ~0.25 mM) (see Note 12). 5. Add 2 equivalents of iodine in DMSO to the peptide solution. The solution becomes yellow indicating that an excess of iodine has been added to the peptide solution. 6. Vortex the solution for 30 s (see Note 13). 7. Then immediately add a few drops of the DTT solution with a Pasteur pipette until the yellow color disappears, indicating that the excess of iodine has been decomposed.

3.1.8 HPLC Purification of SUMO-2/3C and SUMO-2/3N Peptides

1. Purify immediately by semi-preparative HPLC. 2. Gradient used for the HPLC purification of peptide SUMO2N: 0–25% B in 5 min, then 25–35% B in 40 min, flow rate 6 mL/min, 50
C, UV detection at 215 nm.

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3. Gradient used for the HPLC purification of peptide SUMO3N: 0–25% B in 5 min, then 25–35% B in 40 min, flow rate 6 mL/min, 50
C, UV detection at 215 nm. 4. Gradient used for the HPLC purification of peptide SUMO2/3C: 0–20% B in 5 min, then 20–40% B in 40 min, flow rate 6 mL/min, 50
C, UV detection at 215 nm 5. Analyze the collected fractions by analytical UPLC-MS, and pool the pure fractions in a 50 mL plastic tube. 6. Place the tubes in liquid nitrogen to freeze the peptide solutions and lyophilize for 2 or 3 days to obtain the purified SUMO-2N, SUMO-3N, SUMO-2/3C peptide segments (see Note 10). 7. Yield for peptide SUMO-2N: 34.4 mg of crude product furnished 11.2 mg of SUMO-2N, calculated for M (average mass) 5281.17, observed 5281.13 by LC-MS after deconvolution. 8. Yield for peptide SUMO-3N: 30.8 mg of crude product furnished 11.2 mg of SUMO-3N, calculated for M (average mass) 5197.09, observed 5197.17 by LC-MS after deconvolution. 9. Yield for peptide SUMO-2/3C: 31.3 mg of crude product furnished 10.6 mg of SUMO-2/3C, calculated for M (average mass) 5331.82, observed 5331.95 by LC-MS after deconvolution. 3.2 Synthesis of SUMO-2 and SUMO-3

1. Weigh 5.38 mg (1 equiv, 0.825 μmol, 4 mM) of SUMO-xN SEAoff peptide (x ¼ 2 or 3) in a 1.5-mL microfuge tube. 2. Weigh 4.94 mg (1 equiv, 0.825 μmol, 4 mM) of cysteinyl peptide SUMO-2/3C in a 1.5-mL microfuge tube. 3. Weigh 13.42 mg (79.77 μmol, 200 mM final concentration) of MPAA in a 1.5-mL microfuge tube. 4. Weigh 31.6 mg (110 μmol, 200 mM final concentration) of TCEP in a 1.5-mL microfuge tube. 5. Prepare 2 mL of 0.1 M sodium phosphate buffer (pH 7.2)/ 6 M Gn∙HCl. 6. Transfer 1 mL of AcOH glacial, 1 mL of HCl 1 M, and 1 mL of NaOH 6 M in three different 1.5-mL microfuge tubes. 7. Cap all the tubes and transfer them to the glove box under nitrogen atmosphere. 8. Add 0.551 mL of the sodium phosphate buffer from step 5 to the microfuge tube containing TCEP (see step 4). Vortex until dissolution of the TCEP and transfer 0.399 mL of this solution to the microfuge tube containing MPAA (see step 3). Add NaOH 6 M (30 μL) with the micropipette and vortex until dissolution of the MPAA. The final reagent concentration is 181 mM for MPAA and 181 mM for TCEP.

Aspartimide Formation During SEA-Mediated Ligation

25

9. Measure the pH of the solution prepared in step 8 and adjust to pH 5.5 by adding NaOH 6 M or HCl 1 M with the micropipette (2–20 μL). 10. Add 0.205 mL of the solution prepared in step 8 to the microfuge tube containing the SUMO-xN SEAoff peptide (see step 1). Vortex to dissolve the peptide, and then add this solution to the microfuge tube containing the cysteinyl peptide SUMO-2/3C (see step 2). Vortex to dissolve the peptide. 11. Place the reaction tube from step 10 in a thermomixer kept at 25
C with agitation. 12. To monitor the ligation reaction (just after preparing the reaction mixture at step 10 and every 6–7 h until ~33 h of reaction), transfer 1.5 μL of the reaction mixture to a microfuge tube, and quench the reaction by acidifying the sample with 100 μL of 10% aqueous AcOH (vol/vol). Vortex and remove the sample from the glove box. 13. Remove MPAA present in the reaction mixture by extracting the aqueous phase with 1 mL of diethyl ether. Remove the diethyl ether phase with a Pasteur pipette. 14. Repeat step 13 twice. 15. Inject 10 μL of the sample isolated in step 14 into the analytical UPLC-MS system to confirm that the starting peptide segments have been consumed to produce the ligation product SUMO-2 or SUMO-3. 16. After 33 h, remove the reaction tube from the glove box. 17. Transfer the contents of the reaction tube into a 5-mL microfuge tube. 18. Add 0.500 mL of phosphate buffer/6 M Gn∙HCl to the reaction mixture and 70.5 μL of glacial AcOH to a final concentration of 5–10% (vol/vol) with a micropipette to acidify the solution down to a pH of 2–3 (pH paper). 19. Extract the MPAA from the aqueous phase with 2 mL of diethyl ether as done in step 13. Remove the diethyl ether phase with a Pasteur pipette. 20. Repeat step 19 twice. 21. Remove the dissolved diethyl ether by flushing the tube with nitrogen (~2 min). 22. Purify the target SUMO-2 or SUMO-3 proteins using a Waters XBridge BEH300 C18 reverse-phase column (10  250 mm; pore size 300 A˚; particle size: 5 μm) and a semi-preparative HPLC system; gradient: 0% eluent B in eluent A to 20% (vol/vol) eluent B in eluent A over 5 min, and then 20% (vol/vol) eluent B in eluent A to 40% (vol/vol) eluent B in eluent A over 60 min (UV detection at 215 nm). Analyze the

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fractions using the MALDI-TOF mass spectrometer. Mix 1.2 μL of the DHB solution with 1 μL of the sample on the MALDI plate, and let dry at room temperature in air. 23. Pool the pure fractions in a 50-mL plastic tube, freeze the solution with liquid nitrogen, and lyophilize it for 2 or 3 days. The reaction yields 4.20 mg (78% yield) of SUMO2 and 4.46 mg (83% yield) of SUMO-3. 24. Analyze the purified products by UPLC-MS. Acquity UPLC ˚ 1.7 μm 2.1 mm  150 mm, 50
C. peptide BEH C18 300 A Flow 0.400 mL/min, eluent A 0.1% trifluoroacetic acid in water, eluent B 0.1% trifluoroacetic acid in 100% acetonitrile. Gradient from 0% eluent B to 70% eluent B in 20 min. 25. SUMO-2: Calculated for M (average mass) 10477.24, observed 10476.71 after deconvolution. 26. SUMO-3: Calculated for M (average mass) 10393.66, observed 10393.84 after deconvolution.

4

Notes 1. Aspartimide by-product formation can occur during NCL [18]. For example, the assembly of Ras protein (166 amino acid residues) by NCL yielded a product having 18  2 uma less than expected [19]. The mass loss occurred during the last ligation and could be due to a loss of water or ammonia. The site of dehydration or deamination was not identified. For another example, see ref. 20. 2. The solution can be stored or used for up to 2 weeks at room temperature. 3. This solution can be used up to 24 h after preparation. For the automated peptide elongation step, this solution is renewed every day. 4. HATU is a uronium-based coupling reagent which reacts efficiently with amines and thus can cause undesired peptide termination if present in excess. To avoid the presence of unreacted HATU during the coupling step, the amino acid is used in slight molar excess and the amino acid is activated prior to the coupling step. 5. The capping step is mandatory even if the chloranil test is negative, because the sensitivity of the test for the secondary amino group of the SEA resin is lower than for α-amino groups. 6. The synthesis of the ~50-amino-acid SEAoff peptide segments SUMO-xN is performed using the SEA-Trt-ChemMatrix® resin. The synthesis program must consider the large swelling

Aspartimide Formation During SEA-Mediated Ligation

27

volume of the starting SEA-Trt resin and the significant mass increase of the peptidyl resin that occurs during the peptide elongation step. 7. We recommend to confirm the coupling of Fmoc-Asp(OtBu) [Dmb]Gly-OH by cleaving an aliquot in TFA and analyzing the cleaved peptide by MALDI-TOF. 8. MALDI-TOF analysis of the cleaved peptides might show the expected peaks and also products of higher mass (+150 uma) due to the incomplete removal of the Dmb group. 9. We recommend that the volume of the diethyl ether/n-heptane solution be at least 20 times that of the peptide solution in TFA. 10. The peptide solution can be frozen quickly using liquid nitrogen. The tube must be checked before starting the lyophilization because it can crack during this step. 11. Iodine is available as granules. Therefore, weighting a given amount of iodine precisely is difficult to achieve. 12. SUMO-xN SEAoff peptides (x ¼ 2,3) have a limited solubility, and the use of Gn∙HCl, which is a classical additive for improving the solubility of the peptide segments, was mandatory in this case. Iodine oxidation can be performed in the absence of Gn∙HCl and at mM concentrations when solubility is not an issue. 13. The duration of the iodine treatment should not exceed 30 s; otherwise iodine can react with some amino acid residues such as tyrosines. For the same reason, the amount of iodine which is added to the peptide solution must not exceed 2 equivalents. This oxidation procedure is not compatible with the presence of free cysteine thiols on the peptide chain.

Acknowledgments This study was supported by the National Centre for Scientific Research (CNRS), the University of Lille, and the Pasteur Institute of Lille. References 1. Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85:2149–2154 2. Behrendt R, White P, Offer J (2016) Advances in Fmoc solid-phase peptide synthesis. J Pept Sci 22:4–27 3. Voorter CE, de Haard-Hoekman WA, van den Oetelaar PJ, Bloemendal H, de Jong WW (1988) Spontaneous peptide bond cleavage

in aging alpha-crystallin through a succinimide intermediate. J Biol Chem 263:19020–19023 4. Joshi AB, Sawai M, Kearney WR, Kirsch LE (2005) Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon. J Pharm Sci 94:1912–1927 5. Geiger T, Clarke S (1987) Deamidation, isomerization, and racemization at asparaginyl

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and aspartyl residues in peptides. Succinimidelinked reactions that contribute to protein degradation. J Biol Chem 262:785–794 6. Stephenson RC, Clarke S (1989) Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J Biol Chem 264:6164–6170 7. Tyler-Cross R, Schirch V (1991) Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. J Biol Chem 266:22549–22556 8. Suzek BE, Huang H, McGarvey P, Mazumder R, Wu CH (2007) UniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics 23:1282–1288 9. Agouridas V, El Mahdi O, Diemer V, Cargoet M, Monbaliu J-CM, Melnyk O (2019) Native chemical ligation and extended methods. Mechanisms, catalysis, scope and limitations. Chem Rev 12:7328–7443 10. Wucherpfennig TG, Pattabiraman VR, Limberg FRP, Ruiz-Rodrı´guez J, Bode JW (2014) Traceless preparation of C-terminal α-ketoacids for chemical protein synthesis by α-ketoacidhydroxylamine ligation: synthesis of SUMO2/3. Angew Chem Int Ed 53:12248–12252 11. Mulder MPC, Merkx R, Witting KF, Hameed DS, El Atmioui D, Lelieveld L, Liebelt F, Neefjes J, Berlin I, Vertegaal ACO et al (2018) Total chemical synthesis of SUMO and SUMO-based probes for profiling the activity of SUMO-specific proteases. Angew Chem Int Ed 57:8958–8962 12. Ollivier N, Dheur J, Mhidia R, Blanpain A, Melnyk O (2010) Bis(2-sulfanylethyl)amino native peptide ligation. Org Lett 12:5238–5241

13. Johnson EC, Kent SBH (2006) Insights into the mechanism and catalysis of the native chemical ligation reaction. J Am Chem Soc 128:6640–6646 14. Bouchenna J, Se´ne´chal M, Drobecq H, Vicogne J, Melnyk O (2019) Total chemical synthesis of all SUMO-2/3 dimer combinations. Bioconjug Chem 30:2967–2973 15. Bouchenna J, Se´ne´chal M, Drobecq D, Stankovic-Valentin N, Vicogne J, Melnyk O (2019) The role of the conserved SUMO-2/3 cysteine residue on domain structure investigated using protein chemical synthesis. Bioconjug Chem 30:2684–2696 16. Boll E, Drobecq H, Ollivier N, Blanpain A, Raibaut L, Desmet R, Vicogne J, Melnyk O (2015) One-pot chemical synthesis of small ubiquitin-like modifier (SUMO) proteinpeptide conjugates using bis(2-sulfanylethyl) amido peptide latent thioester surrogates. Nat Protoc 10:269–292 17. Boll E, Drobecq H, Ollivier N, Raibaut L, Desmet R, Vicogne J, Melnyk O (2014) A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1 peptide conjugate. Chem Sci 5:2017–2022 18. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779 19. Becker CF, Hunter CL, Seidel R, Kent SBH, Goody RS, Engelhard M (2003) Total chemical synthesis of a functional interacting protein pair: the protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1. Proc Natl Acad Sci U S A 100:5075–5080 20. Cowper B, Shariff L, Chen W, Gibson SM, Di W-L, Macmillan D (2015) Expanding the scope of N ! S acyl transfer in native peptide sequences. Org Biomol Chem 13:7469–7476

Chapter 3 Using the Interactive Tool of the Protein Chemical Synthesis Database Vangelis Agouridas and Oleg Melnyk Abstract Over the last 25 years, chemoselective amide bond-forming reactions have established themselves as an essential tool for the total chemical synthesis of peptides and proteins. This spectacular development is echoed in an abundant literature that we have compiled in a database: the Protein Chemical Synthesis (PCS) Database (http://pcs-db.fr). The PCS website provides an interactive tool with a user-friendly interface to get introduced to the most routinely used ligation methods including their scope. It can also be used for simply getting an overview or a track of the most recent advances made in the field of peptide and protein synthesis by means of chemoselective ligation reactions. The aim of this protocol article is to present the content of the database and showcase a typical query with the interactive web interface. Key words Chemical protein synthesis, Chemoselective amide bond-forming reaction, Interactive database

1

Introduction During the last 25 years, the scope of peptides and proteins amenable to chemical synthesis has been considerably extended with the advent of chemoselective amide bond-forming reactions, recently pushing the size of fully synthetic and functional proteins up to 450 residues [1]. Basically, these reactions consist in the chemoselective formation of a native peptide bond between two unprotected peptide segments under mild conditions (Fig. 1) [2]. The Native Chemical Ligation (NCL [3]), a ligation method based on the reaction of a peptide thioester with a cysteinyl peptide, has been extensively used since its discovery in 1994 (Fig. 1a). Since then, some variations have been introduced to expand its scope by modifying the nature of the acyl donor (i.e., hydrazides [4], benzimidazolinones [5], or O,S-, N,S-, or N,Se-acyl shift systems [6]), of the thiol component (thiol or selenol amino acid surrogates [7], auxiliaries [8]), or both (diselenide selenoester ligation, DSL [9],

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Fig. 1 General principle of most commonly used chemoselective amide bond-forming reactions

Fig. 1c). Besides, other mechanistically unrelated ligation methodologies were developed such as the α-ketoacid hydroxylamine ligation (KAHA, Fig. 1d) [10], the serine-threonine ligation (STL, Fig. 1e) [11], and the thioester hydroxamate ligation (Fig. 1f) [12]. Finally, the protein chemist synthetic toolbox has also been

Using the Interactive Tool of the PCS-db

31

enriched by a repertoire of post-ligation reactions that allow for further modifications on assembled proteins (e.g., desulfurization [13]). Since 1994, all of these methods have been used alone or combined in complex reaction schemes to prepare hundreds of proteins. The “Protein Chemical Synthesis Database” [14] (pcsdb.fr) is a comprehensive database that was created in order to facilitate the collection or the retrieval of information about the synthetic design of proteins (see Note 1). Moreover, the PCS-db proves a particularly powerful tool when it comes to put the domain in perspective or compare where a specific method stands in relation to the other ones for a specific application.

2

Presentation of the PCS Database: Conceptual and Logical Design The PCS-db was built by collecting different types of descriptors from more than 600 articles reporting the chemical synthesis of proteins by means of chemoselective amide bond-forming reactions since 1994. Only targets of biological significance were retained, whereas model peptides used for methodological studies, polymers, or hybrid materials were systematically discarded. The first level of information available for registered peptides and proteins concerns their inherent characteristics: name, year of publication, and length. The second level of information is relative to their synthetic design. It provides some details about the type of ligation chemistry used and the number of ligations achieved to assemble the target proteins but also the nature of the junction residues, the use of amino acid surrogates, thiol auxiliaries, and/or the application of postligation treatments (see Note 2). All the abovementioned data were compiled in a table file which was processed with a cloud-based self-service usually used for data management in business intelligence. A copy of the table in MS excel format remains available in the download section of the PCS-db website, though with a limited number of functionalities. The web interface of the PCS-db provides a user-friendly tool where queries are simply made by mouse-clicking buttons which activate/deactivate various filters and display refined subsets of the collected data in a table. Additionally, a “graphical overview” module (PCS-GO) complements the database. The PCS-GO module is composed of interactive charts based on the PCS-db dataset that visitors can manipulate at their convenience to quickly get a synoptic view of the domain. Finally, the PCS website also features a full page dedicated to instructions which provides detailed information on the meaning of each filter and a bibliographical page presenting a selection of landmark books, reviews, and papers to get introduced to the main concepts of chemoselective peptide ligation reactions.

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Materials The PCS website (pcs-db.fr) proposes a dynamic, interactive, and intuitive environment that requires no particular computer skills. It is accessible from any personal computer, tablet, or mobile phone connected to the Internet with an up-to-date web browser (see Notes 3, 4 and 5). The very basic examples below detail the procedure for making queries on the PCS-db (update of March 25, 2019: 931 entries which correspond to the total number of proteins available in this version of the database). Of course, all search criteria can be combined at will in more complex scenarios (see Note 6).

4

Methods

4.1 Scenario 1 (Use of the PCS-db Module)

In scenario 1, one would like to design a synthetic approach for a protein of 180 residues with the following constraints: (1) the protein will be produced chemically, without resorting to recombinant technologies; (2) the exclusive use of NCL or NCL-derived methods is required; and (3) a Q-C (Gln-Cys) junction will be assembled by ligation. How can the PCS-db help him/her retrieve works from the literature responding to this query? 1. Go to the “pcs-db.fr” website. On the homepage, select the “PCS-DB” menu to display the PCS-db control panel (Fig. 2).

Fig. 2 General control panel of the Protein Chemical Synthesis Database (PCS-db)

Using the Interactive Tool of the PCS-db

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2. [OPTIONAL] On the bottom-right corner of the interactive table, click the double-headed arrow to toggle full screen mode. 3. To have a relevant answer set regarding the size of your target, search for proteins whose length is comprised between 160 and 200 residues using the numeric range slicer (Fig. 2, step 1). Application of this first search criterion results in a significant shrinking of the answer set (from 931 to 41 answers). 4. Exclude the recombinant proteins by selecting “No” in the “EPL filter” box (Fig. 2, step 2). Thirty-four entries remain available in the database. 5. The answer set can still be refined according to the initial search criteria. In the “type of ligation” menu, select all NCL and NCL-extended methods still available (i.e,. NCL, hydrazides, N,S-acyl shift, N,Se-acyl shift, O,S-acyl shift, Nbz, DSL) (Fig. 2, step 3). Removal of the KAHA ligation which is not a thiolthioester exchange-based reaction discards two additional references. 6. In the “C-terminal Residue Control Panel,” click Q to select synthetic works which describe the assembly of at least one Q-C junction (Fig. 2, step 4). 7. Check the result in the bottom table. You are now redirected to seven entries describing either the total chemical synthesis of the NK1 domain of the human hepatocyte growth factor or the synthesis of various EPO glycoforms. The assembly of these proteins is described in five different publications whose abstracts can be directly accessed by clicking the link right next to the reference whenever available (Fig. 2, step 5). 8. If needed, the answer set can still further be refined by mouseclicking the unexploited descriptors. As an example, consider that the total synthesis of the targeted protein is finally to be conducted on a solid support, clicking on the solid phase button will point to only one publication discussing the production of biotinylated NK1 (Fig. 2, step 6) [15]. 9. To reset all filters at once and make a new query, click the “clear filters” image (Fig. 2, step 7). 4.2 Scenario 2 (Use of the PCS-GO Module)

The PCS-GO module is a particularly powerful tool to generate statistical overviews of the domain either for enriching a course material or for illustrating an oral presentation. The following example shows how to use it. The objective here is to evaluate the occurrence of synthetic designs involving three or more ligation reactions to assemble a protein. 1. Go to the “pcs-db.fr” website. On the homepage, select the “PCS-GO” menu to display the PCS-GO control panel (Fig. 3). The PCS-GO module is composed of various

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Fig. 3 Interface of PCS-GO module

graphical elements which represent the number of peptides and proteins produced each year by means of chemoselective ligation reactions (Fig. 3a), provide quantitative information about the refined datasets (Fig. 3b), or are informative of the number or the type of ligation reactions used to assemble proteins (Fig. 3c, d). The use of amino acid surrogates, auxiliaries, or desulfurization approaches is reported in Fig. 3e. 2. [OPTIONAL] On the bottom-right corner of the interactive table, click the double-headed arrow to toggle the full screen mode. 3. In rectangle C, left-click on the area corresponding to three ligation reactions. While holding down the Ctrl key, left-click the areas corresponding to 4, 5, 6, and 8 ligations (Fig. 3, step 1). 4. Other graphical representation instantly refreshes to deliver adjusted statistics that allow to appreciate the importance of multisegment design over the recent years.

5

Notes 1. The PCS database can be cited with mention of our 2017 publication in Bioorganic and Medicinal Chemistry (see ref. 14).

Using the Interactive Tool of the PCS-db

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2. More comprehensively, the PCS-db interactive table can filter entries according to year of publication, type or number of ligations, author names, or length of proteins. It can also display results according to whether or not synthetic targets have been produced using recombinant technologies, solidphase synthesis, microfluidics, use of amino acid surrogates, auxiliaries, or post-ligation treatments such as desulfurization. It is also possible to quickly visualize all peptides or proteins whose synthesis has involved a ligation step at a particular junction residue. Finally, an interactive plot chart located at the center of the interface can be clicked to select one particular entry in the database. Each point represents one or more proteins as a function of its length and the number of ligation steps required to assemble it. 3. The language of the PCS-db module is set to be the default language of the web browser used to display the database. Language can be changed by modifying the language settings of your web browser. 4. Https traffic, which is the kind of protocols used for the PCS-db, might be blocked by firewalls. In such a case, an error message is displayed and the database will not load. The problem can be solved by changing the site permission parameters of your firewall or asking your computer and networkrelated service to allow access to the PCS-db website at your institution. 5. Other tools have been recently developed in order to facilitate the identification of targets of interest or to assist the protein chemist in the synthetic design of peptides and proteins: the ProteoFind script [16] and the Alligator software [17], respectively. 6. In upcoming PCS updates, new options will be added that will enable to filter results according to whether or not synthesized peptides or proteins are cyclic, possess post-translational modifications, or feature tags (peptidic or non-peptidic such as fluorophores) [18]. Besides, the content of the database will be regularly updated.

Acknowledgments This work was supported by the Centre National de la Recherche Scientifique (CNRS), the University of Lille, and Institut Pasteur de Lille.

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References 1. Sun H, Brik A (2019) The Journey for the Total Chemical Synthesis of a 53 kDa Protein. Acc Chem Res 52:3361–3371 2. Agouridas V, El Mahdi O, Diemer V, Cargoe¨t M, Monbaliu JCM, Melnyk O (2019) Native chemical ligation and extended methods. Chem Rev 119:7328–7443 3. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779 4. Fang GM, Li YM, Shen F, Huang YC, Li JB, Lin Y, Cui HK, Liu L (2011) Protein chemical synthesis by ligation of peptide hydrazides. Angew Chem Int Ed 50:7645–7649 5. Blanco-Canosa JB, Dawson PE (2008) An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew Chem Int Ed 47:6851–6855 6. Melnyk O, Agouridas V (2014) From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N, S-acyl or N,Se-acyl migration reactions. Curr Opin Chem Biol 22:137–145 7. Wong CTT, Tung CL, Li X (2013) Synthetic cysteine surrogates used in native chemical ligation. Mol Biosyst 9:826–833 8. Burke HM, McSweeney L, Scanlan EM (2017) Exploring chemoselective S-to-N acyl transfer reactions in synthesis and chemical biology. Nat Commun 8:15655 9. Mitchell NJ, Malins LR, Liu X, Thompson RE, Chan B, Radom L, Payne RJ (2015) Rapid additive-free selenocystine-selenoester peptide ligation. J Am Chem Soc 137:14011–14014 10. Bode JW, Fox RM, Baucom KD (2006) Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and

a-ketoacids. Angew Chem Int Ed 118:1270–1274 11. Zhang Y, Xu C, Lam HY, Lee CL, Li X (2013) Protein chemical synthesis by serine and threonine ligation. Proc Natl Acad Sci U S A 110:6657–6662 12. Dunkelmann DL, Hirata Y, Torato KA, Cohen DT, Zhang C, Gates ZP, Pentelute BL (2018) Amide-forming chemical ligation via O-acyl hydroxamic acids. Proc Natl Acad Sci U S A 115:201718356 13. Wan Q, Danishefsky SJ (2007) Free-radicalbased, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew Chem Int Ed 46:9248–9252 14. Agouridas V, El Mahdi O, Cargoe¨t M, Melnyk O (2017) A statistical view of protein chemical synthesis using NCL and extended methodologies. Bioorg Med Chem 25:4938–4945 15. Ollivier N, Desmet R, Drobecq H, Blanpain A, Boll E, Leclercq B, Mougel A, Vicogne J, Melnyk O (2017) A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins. Chem Sci 8:5362–5370 16. Shigenaga A, Naruse N, Otaka A (2018) Proteofind: a script for finding proteins that are suitable for chemical synthesis. Tetrahedron 74:2291–2297 17. Jacobsen MT, Erickson PW, Kay MS (2017) Aligator: a computational tool for optimizing total chemical synthesis of large proteins. Bioorg Med Chem 25:4946–4952 18. Agouridas V, El Mahdi O, Melnyk O. Protein chemical synthesis in medicinal chemistry. Manuscript in preparation.

Chapter 4 Only a “Click” Away: Development of Arginine-Rich Peptide-Based Materials Using Click Chemistry Mariana Barbosa, Fabı´ola Costa, Ca´tia Teixeira, M. Cristina L. Martins, and Paula Gomes Abstract The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) coupling is one of the most interesting chemoselective reactions that match the “click chemistry” concept. Both azide and alkyne moieties, easily incorporated into polymers and biomolecules, react selectively with each other yielding a stable triazole link. Thus, CuAAC “click” reaction has been widely explored as a powerful tool for the covalent linkage of biomolecules. In this context, this highly efficient and regioselective chemistry was selected for covalently immobilize arginine-rich peptides onto chitosan, a biopolymer with interesting bioactive properties. This chapter focuses on experimental procedures for immobilization of arginine-rich peptides by means of the aforementioned CuAAC reaction. Peptide grafting onto chitosan via CuAAC either on (1) ground chitosan powder (type 1 conjugation) or (2) preformed chitosan thin films (type 2 conjugation) is described, demonstrating the versatility of the selected coupling reaction. In addition, given its relevance in this particular context, solid-phase peptide synthesis of alkyne-modified peptides and conversion of chitosan amines into azides are also described. Overall, this work is intended to convey useful guidelines for the immobilization of arginine-rich peptides onto amine-functionalized polymers, by means of click chemistry via CuAAC. Key words Arginine, Azide-alkyne coupling, Chitosan, Click chemistry, Huisgen’s 1,3-dipolar cycloaddition, Peptide, Polymer, Tethering, Bioconjugation, Peptide-based materials

1

Introduction Highly efficient chemistries toward development of tailor-made materials for specific applications have been widely explored in recent years, mostly aimed at creating new biomaterials with potential application in biomedical engineering. The design and production of highly functional and specific biomaterials has been mainly driven by the need to enhance the therapeutic effectiveness of tissue regeneration approaches. Cells in vivo are enclosed in a threedimensional (3D) extracellular matrix (ECM) composed of numerous biochemical and mechanical cues capable of modulating

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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cellular behavior, while the fibrous protein structures of the ECM confer mechanical support. Cell signaling molecules, such as adhesion molecules and growth factors, are also part of the ECM composition [1–3]. Hence, knowing the organization and roles of the ECM is of vital importance for creating novel biomimetic and bioinstructive materials as cell culture scaffolds. These 3D platforms should be designed to mimic the ECM milieu and promote cell-matrix interactions responsible for regulating cellular activity and consequent tissue organization and remodeling [1–3]. However, incorporating specific ECM cell signaling molecules, or other relevant bioactive molecules, into these scaffolds remains a major challenge. Currently, prolonged biological activity of functionalized biomaterials is thwarted by stability problems after in vivo administration as, in most cases, bioactive molecules are simply adsorbed to a scaffold material or tethered through ineffective chemical strategies [2, 3]. In fact, successful tethering of bioactive molecules onto materials not only depends on the material receptiveness for functionalization, but also on how modification strategies will influence the properties of both the material and the tethered molecule. For these reasons, it is important to explore strategies providing easy functionalization of polymers with building blocks with relevant biological activity and tunable physicochemical characteristics to produce novel biomimetic materials. In this context, peptide domains have been incorporated into polymeric matrices in order to obtain highly multifunctional biomaterials with controllable structure and unique responsive properties. Nowadays, the significative improvements on both synthesis and characterization methods boosted the biomedical application of peptide-based materials [1, 4–6]. The role of bioactive peptides in biomedical research and drug development has been increasing over the years [7, 8]. In fact, peptides have found applications in a wide range of therapeutic areas acting as growth factors, immunomodulators, or even as antimicrobial agents [7, 9–14]. Most physiological processes, such as cellular function and intercellular communication, are regulated by peptides. The particular amino acid sequences of peptides or proteins affect the interaction mechanisms behind the molecular recognition and binding affinity between peptides/proteins and ligands, which confers them highly specific biological effects [15, 16]. Peptides, particularly those having short sequences, are usually biocompatible and are not associated with acute immune responses, as they are composed of naturally occurring or metabolically degradable amino acids [17–20]. Most of peptide drug candidates are similar to its bioactive parent molecules, which significantly reduces the risk of unpredicted side reactions. Also, its smaller

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size reduces the production complexity and costs [16, 21]. Moreover, peptides can be tailored to meet specific properties by simple modification approaches [20]. Hence, it comes with no surprise that the number of peptide-based drugs submitted to clinical trials is progressively increasing [9, 21]. In this context, peptides can be used either as the bioactive component itself or in the modification of several other molecules or biomaterials [21, 22]. Consequently, the chemical synthesis of peptides continues to be an important and growing area [17, 23]. Clinical and therapeutic applications of peptides are, however, limited by some of their physicochemical characteristics. Their large molecular weight and high hydrophilic behavior limit their diffusion across the membrane cell layers and consequently lower their bioavailability. Peptides are also associated with low metabolic stability, even if administered by parenteral routes, as they are susceptible to proteolytic degradation and can be readily eliminated from the body [24, 25]. Their short half-life in vivo can be overcome by increasing the administration concentration, but this can lead to increased toxicity profiles [24, 26]. Thus, development of peptidebased compounds is hindered by these stability issues [16, 27]. Therefore, strategies to improve the delivery routes and extend the bioactivity of therapeutic peptides in vivo are essential to foster their application in the biomedical field [16, 27]. An optimized delivery matrix should ensure a controlled and sustained administration at the desired target site, assuring a correct dose and avoiding recurrent administrations throughout the treatment. Furthermore, a suitable peptide delivery carrier can prevent peptide degradation [27, 28]. Noteworthy, the site-specific peptide delivery systems help to bypass many of their bioavailability and toxicity issues [29, 30]. In this connection, biocompatible polymers are being used as important peptide delivery systems carriers, due to their versatility, as they can be tailored and altered to meet the requirements for a specific application, and their physicochemical characteristics. Polymers can be easily modified by changing their molecular components or the polymerization conditions, or by functionalizing with adequate functional groups for subsequent chemoselective peptide tethering [28, 29, 31]. This fine-tuning process needs to take into account several aspects, namely, the peptide loading capacity and subsequent release profiles, and the exposure and distribution of the tethered bioactive peptide. Moreover, it is imperative to evaluate if the peptide immobilization will affect the biological activity of both polymer and peptide [32]. The concept of “click” chemistry reactions has emerged along with the twenty-first century, as a highly promising strategy for chemoselective conjugation between two building blocks, including peptide grafting onto other moieties [33]. The term “click” highlights the easy and selective fashion in which two chemically dissimilar functional groups react exclusively with each other, under

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Fig. 1 The Huisgen 3-dipolar cycloaddition between azides and alkynes: (a) uncatalyzed thermal azide-alkyne cycloaddition yielding a mixture of the 1,5and the 1,4-triazole regioisomers, and (b) Cu(I) catalysis in alkyne-azide coupling reactions favoring exclusive formation of the 1,4-regioisomer

mild conditions, even in the presence of other reactive functional groups and molecules. In other words, “click” reactions are fast, stereospecific, modular, and orthogonal. So, the range of applications of “click” chemistry in the field of biomedical research has been steadily increasing [34–36]. Although there are many reactions that fit the “click” chemistry concept, the reaction between alkynes and azides, i.e., the Huisgen’s 1,3-dipolar cycloaddition, affording stable triazoles (Fig. 1), is the one that reached higher popularity for the most diverse reported applications of “click” chemistry. Both azide and alkyne groups involved react exclusively with each other and can be readily incorporated into several distinct molecules [37–40]. Also, the triazole ring formed between the two functional groups is usually biocompatible, extremely stable, and [bio-]chemically inert to most conditions and environments [38, 39]. Interestingly, diverse biological effects of potential therapeutic interest, such as antiretroviral and antimicrobial activities, have been assigned to triazoles [41]. In most reported cases, the Huisgen’s 1,3-dipolar cycloaddition reaction is carried out under Cu(I) catalysis, as this occurs effectively and regioselectively in an extensive range of mild aqueous environments and with distinct Cu(I) sources. The most commonly utilized in these copper-catalyzed azide-alkyne cycloadditions (CuAAC) are low-cost Cu(II) salts (e.g., sulfate pentahydrate, acetate). These are used in combination with sodium ascorbate that acts as an in situ reducing agent to convert Cu (II) into Cu(I) [39, 42]. CuAAC reactions have been extensively studied for the synthesis of copolymers and for the grafting of bioactive molecules, such as peptides, to polymers. In this latter case, the peptide has to be firstly modified with either an azide or an alkyne group, both of which are stable to standard peptide synthesis protocols [40, 43].

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Fig. 2 Formation of arginine-DHA adduct resultant from the reaction between the guanidinium group of arginine side chain with DHA (the oxidation product of ascorbate)

The aforementioned remarkable characteristics of CuAAC reactions, namely, chemoselectivity and straightforward experimental conditions, prompted a plethora of new tissue engineering applications. The mild aqueous reaction media usually employed in CuAAC are not only sustainable and ecofriendly but also compatible with development of biomaterials for cell culture and also with drug release materials [44]. However, for the particular case of immobilizing peptides via CuAAC, one cannot forget the peculiar characteristics of peptides, highly dependent on their specific sequences and content on given amino acid residues. For instance, ascorbic acid and derived salts commonly used in CuAAC cause side-chain modification of arginine residues (Fig. 2). Ascorbate, used to reduce Cu(II) to Cu(I) as previously stated, originates dehydroascorbic acid (DHA) as the resultant oxidation product, which has been reported to react with the side chain of arginine residues, which hampers CuAAC-mediated grafting of argininerich peptides [45]. In such cases, one could try to bypass this limitation, by avoiding use of the in situ reducing agent, i.e., using Cu(I) salts; however, these are significantly more toxic, expensive, and air/moisture-sensitive than their Cu (II) counterparts [8, 46, 47]. In view of this, we have developed a protocol to graft arginine-rich peptides via CuAAC onto chitosan, either as ground bulk powder or as a preformed ultrathin film, which can be applied to other biopolymers allowing for insertion of azide functional groups. Noteworthy, to the extent of our knowledge, our group was pioneer in grafting peptides onto bulk ground chitosan, as peptide grafting is typically carried out onto pre-assembled surfaces or scaffolds. In this later case, different materials mean that immobilization must be repeated, and experimental procedures have to be optimized again. On the other hand,

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powdered biopolymers functionalized with peptides, as those that can be prepared using our protocol, are suitable for scale-up and development of diverse materials. Notwithstanding, the versatility of the CuAAC chemistry herein described is demonstrated by the fact that it can also be successfully employed for peptide tethering onto previously assembled scaffolds like, e.g., chitosan ultrathin films.

2

Materials

2.1 Synthesis of the Designed Peptide Sequences

l

l

2.2 Conversion of Chitosan Amine Groups into Azide Groups

2.3 Peptide-Chitosan Conjugation Via CuAAC

N-(9-fluorenylmethoxycarbonyl)-protected Rink amide MBHA resin (Fmoc-Rink-MBHA), 100–200 mesh, 0.38 mmol/g loading. Nα-Fmoc-protected amino acids, including N-protected non-natural 6-aminohexanoic acid (Fmoc-Ahx-OH) and propargylglycine (Fmoc-Pra-OH).

l

2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU).

l

Piperidine.

l

N,N-Dimethylformamide (DMF).

l

N-Methylpyrrolidone (NMP).

l

N-Ethyl-N,N-diisopropylamine (DIPEA).

l

1-Hydroxybenzotriazole (HOBt).

l

Trifluoroacetic acid (TFA).

l

Triisopropylsilane (TIS).

l

High molecular weight chitosan, 94% degree of deacetylation (DD).

l

Dialysis membrane (MW cutoff 3.5 kDa).

l

Potassium carbonate (K2CO3).

l

Imidazole-1-sulfonyl azide hydrochloride (ISA·HCl).

l

Modified peptides from 2.1.

l

Modified chitosan from 2.2.

l

Dialysis membrane (MW cutoff 3.5 kDa).

l

Type 1 water (ultrapure water with resistivity superior to 18 MΩ·cm, conductivity inferior to 0.056 μS/cm, total organic carbons below 5 ppb, and bacterial count inferior to 1 CFU/ mL) (see Note 8).

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3

43

l

Type 2 water (resistivity greater than 1 MΩ·cm, conductivity and total organic carbons under 1 μS/cm and 50 ppb, respectively, and bacteria concentration inferior to 100 CFU/mL).

l

Sodium ascorbate.

l

Copper(II) sulfate pentahydrate (CuSO4·5H2O).

l

N-Ethyl-N,N-diisopropylamine (DIPEA).

l

Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA).

l

Ethylenediaminetetraacetic acid (EDTA).

l

Aminoguanidine hydrochloride.

Methods

3.1 Synthesis of the Designed Peptide Sequences

The antimicrobial peptide, Dhvar-5 [48–51], was taken as the model arginine-rich peptide. Conveniently modified Dhvar-5 derivatives were designed to (1) be modified at either the N- or the C-terminus with propargylglycine (Pra), a non-natural amino acid bearing a side-chain alkyne, and (2) have a flexible 6-carbon spacer (6-aminohexanoic acid; Ahx) between Pra and the bioactive Dhvar5 sequence (Fig. 3). Peptides were assembled by standard 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) solid-phase peptide synthesis (SPPS) assisted with microwave (MW) energy, using a Liberty1 Microwave Peptide Synthesizer from CEM Corporation (Matthews, NC, USA). For the synthesis of the N-modified Dhvar-5 derivative (Fig. 3b), detailed procedures were as follows: 1. Fmoc-Rink-MBHA resin was preconditioned for 15 min in DMF and then transferred into the MW-reaction vessel. 2. The initial Fmoc removal step was carried out using 20% piperidine in DMF containing 0.1 M of HOBt in two MW irradiation pulses: 30 s at 24 W plus 3 min at 28 W, in both cases with maximum temperature allowed no higher than 75
C (see Note 1).

Fig. 3 Synthesized derivatives of the antimicrobial peptide Dhvar-5, modified with a spacer and an alkyne functionality at either the peptide’s (a) C-terminus or (b) N-terminus

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3. The C-terminal amino acid of the Dhvar-5 sequence was next coupled to the deprotected resin, using 5 molar equivalents (eq) of the respective Fmoc-protected building block in DMF (0.2 M), 5 eq of 0.5 M HBTU/HOBt in DMF, and 10 eq of 2 M DIPEA in NMP; the coupling step was carried out for 5 min at 35 W MW irradiation, with maximum temperature allowed no higher than 75
C. 4. The remaining Fmoc-protected amino acids were sequentially coupled in the C ! N direction by means of similar deprotection (Fmoc removal) and coupling cycles, until the sequence of Dhvar-5 was completed. 5. For the additional coupling of the Ahx spacer and the N-terminal Pra, manual Fmoc/tBu SPPS was employed (see Note 2). The synthesis of the C-terminally modified Dhvar-5 peptide derivative (Fig. 3a) was carried out by similar methods, starting with manual incorporation of Pra, followed by Ahx, onto the resin. Then, the resulting peptidyl-resin was placed in the MW reaction vessel and the full Dhvar-5 sequence assembled as described above (steps 1–4). Following completion of the sequence assembly, the peptide was released from the resin with concomitant removal of side-chain protecting groups, by a 1.5 h acidolysis at room temperature using a TFA-based cocktail containing TIS and water (95:2.5:2.5 v/v/v) as carbocation scavengers. 3.2 Synthesis of Peptide-Chitosan Conjugates Via CuAAC 3.2.1 Preparation of Ground Azido-Chitosan (N3-Chitosan)

Chitosan was functionalized by direct conversion of the polymer’s amines into azides [52]. Briefly: 1. 100 mg of previously purified chitosan was suspended in 20 mL of type 2 water (see Note 3). 2. 20.5 mg of potassium carbonate and 55.1 mg of ISA·HCl were added to the chitosan suspension, and the mixture left under magnetic stirring for 24 h, at room temperature. 3. The mixture was freeze-dried to afford N3-chitosan (see Note 4).

3.2.2 Synthesis of Peptide-Chitosan Conjugates

The conjugation between the modified arginine-rich peptides and N3-chitosan was obtained under standard CuAAC reaction conditions, i.e., in the presence of Cu(II) sulfate pentahydrate and sodium ascorbate, for in situ generation of the Cu(I) catalyst, in aqueous medium (see Note 5). 1. 100 mg of N3-chitosan was suspended in 20 mL of type 2 water. 2. To the previous suspension, 250 mg of alkyne-peptide and 375.9 mg of aminoguanidinium chloride were added.

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3. Then, 17.2 mg of CuSO4.5H2O, 295.5 mg of THPTA, and 711.4 mg of sodium ascorbate were added to the above suspension. 4. The slurry was left under magnetic stirring, at room temperature, for 48 h. 5. The solid fraction was collected after centrifugation for 10 min at 790 rcf and thoroughly washed with 0.1 M aqueous EDTA, 5% aqueous sodium bicarbonate, and finally type 1 water. 6. The material obtained was dialyzed against 1% hydrochloric acid in type 1 water for 3 days and then against type 1 water for 2 days. 7. The final conjugate (peptide-N3-chitosan) was collected after centrifugation (at 790 rcf for 10 min) and freeze-dried. 3.2.3 Capping of Unreacted Azide Groups

To cap unreacted azide groups, the peptide-N3-chitosan conjugate was further reacted with propargylamine (see Note 6), under CuAAC conditions similar to those described above, in Subheading 3.2.2 (see Note 7): 1. 100 mg of peptide-N3-chitosan was suspended in 20 mL type 2 water. 2. To the above suspension, 3.30 mL of propargylamine, 17.2 mg of CuSO4·5H2O, and 711.4 mg of sodium ascorbate were added. 3. The mixture was left under magnetic stirring, at room temperature, for 48 h. 4. The resulting solid (peptide-chitosan) was washed as above described in steps 4 and 5 of Subheading 3.2.2 and then centrifuged and freeze-dried.

3.3 Synthesis of Peptide-Chitosan Ultrathin Films 3.3.1 Preparation of Chitosan Ultrathin Films

Chitosan ultrathin films were produced according to the work of Barbosa et al. [53], using previously purified chitosan. Briefly: 1. A 50 μL drop of chitosan (0.4% w/v in 0.1 M aqueous acetic acid) was deposited by spin-coating on top of gold substrates (1  1 cm2). 2. Double-layer chitosan ultrathin films were prepared by performing twice the spin-coating process. 3. The chitosan ultrathin films were next neutralized with 0.1 M aqueous NaOH and rinsed with type 1 water (see Note 8). 4. Finally, the films were dried with a gentle stream of argon and stored in sealed plastic Petri dishes saturated with argon until further use.

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3.3.2 Conversion of Chitosan Ultrathin Films’ Amines into Azides

Conversion of amine groups in chitosan ultrathin films into azides was accomplished using an adaptation of the previously optimized method described in Subheading 3.2.2. In brief: 1. Chitosan ultrathin films were covered with an aqueous solution (type 2 water) containing ISA·HCl (2 mM) and potassium carbonate (1.5 mM) and left under orbital shaking at 100 rpm for 24 h, at room temperature. 2. The modified films were rinsed with type 1 water, immersed for 1 min on an ultrasound bath, and rinsed again with type 1 water.

3.3.3 Peptide Tethering onto Functionalized Ultrathin Films

Immobilization of synthesized peptides onto N3-chitosan ultrathin films was obtained under standard CuAAC reaction conditions between the alkyne group of the terminal propargylglycine residue and the azide group on the films, by an adaptation of a previously described method [53]. Procedure was as follows: 1. N3-chitosan ultrathin films were incubated with excess peptide (10 mg/mL) in the presence of aminoguanidinium chloride (0.1 M), in type 2 water. 2. CuSO4.5H2O (2 mM), ligand THPTA (0.01 M), and sodium ascrobate (0.1 M) were added, and the reaction allowed to proceed for 24 h at 37
C, under orbital shaking at 100 rpm. 3. The modified films were sequentially rinsed with type 1 water, 0.1 M aqueous EDTA, 5% aqueous sodium bicarbonate, and again with type 1 water, with a short sonication pulse between each rinsing step. 4. Each sample was finally dried with a gentle stream of argon and stored in plastic Petri dishes saturated with argon.

4

Notes 1. The SPPS assisted with MW energy is widely accepted to increase reaction rates and to improve the synthesis of long or more complex peptide sequences. However, there is also evidence that MW may contribute to undesired side reactions, such as racemization or aspartimide formation, among others. Hence, to overcome such secondary reactions, we recommend adding HOBt to piperidine for Fmoc deprotection [54]. 2. The coupling of selected spacer (Ahx) and alkyne (Pra) moieties should be performed following the manual synthesis strategy. Using the microwave-assisted peptide synthesizer failed to yield the full designed peptides. 3. After purification, chitosan was first functionalized by direct conversion of the polymer’s amines into azides, by means of the diazo-transfer reagent ISA·HCl. The choice of this reagent was

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based not only on its reported efficacy, but also and especially, because it is a green chemistry approach for the conversion of amines into azides, as the reaction can be carried out in water, only requiring the presence of a mild base like K2CO3 [52]. Since chitosan does not dissolve in alkaline medium, the reaction was carried out under heterogeneous conditions. For production of azido-chitosan (N3-chitosan), ISA·HClmediated conversion of amines into azides was carried out both in water, as preferred solvent, and methanol, for comparison. In methanol, only low conversion rates were detected. This finding could be attributed to only partial dissociation of the base, K2CO3, under these non-aqueous conditions, which would result in incomplete neutralization of the diazo-transfer reagent (used as a hydrochloride salt) and protonation of the more basic amine, with the consequent reduction of efficiency. On the other hand, when the reaction was carried out in water, extensive conversions were obtained. 4. Freeze-drying was preferred to drying in a vacuum oven, as the latter causes the ground material to form a stiff agglomerate instead of a powdered product similar to the unmodified chitosan. The stiff agglomerate is difficult to handle and limits subsequent characterization techniques, such as Fourier transform infrared spectroscopy or X-ray photoelectron spectroscopy. 5. To prepare the target peptide-chitosan conjugates, different conditions were tested in order to optimize coupling of arginine-rich peptides with N3-chitosan in aqueous medium containing the Cu2+/ascorbate pair, for in situ formation of the Cu+ catalyst required in CuAAC. As shown by data on Table 1, Table 1 Optimization of CuAAC reaction conditions Reaction conditions Entry Activator

Base Ligand –

Additives

Results

Reference

Sodium ascorbate

Minor conversion detected

[58]

No conversion detected

[42, 52]

Reasonable conversion; final conjugate with less arginines than expected

[55]

1

CuSO4·5H2O –

2

CuBr

3

CuSO4·5H2O –

THPTA

Sodium ascorbate

4

CuSO4·5H2O –

THPTA

Sodium ascorbate, Success of the reaction aminoguanidine hydrochloride

DIEA 2,6-Lutidine Sodium ascorbate

[47, 56]

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the target conjugate could only be obtained when both THPTA, a ligand that stabilizes Cu(I) in solution [55], and excess aminoguanidine hydrochloride, which avoids modification of Arg side chains by ascorbate [56], were employed (entry 4). 6. The capping step of unreacted azide groups, under CuAAC conditions, can be performed immediately after the conjugation between the modified arginine-rich peptides and N3-chitosan and subsequent washing steps, namely, after step 5 of Subheading 3.2.2. Nonetheless, we found that it is important to monitor the reactions by Fourier transform infrared spectroscopy (FT-IR) in order to confirm that the chitosan derivative N3-chitosan polymer is successfully coupled to alkyne-AMP. Hence, we recommend to freeze-dry the final conjugate (peptide-N3-chitosan), as the presence of water in the sample may overlap with important spectral bands, and proceed to FT-IR analysis before moving on to the capping step. 7. As some azide groups remained unreacted, these were capped through a second “click” step using excess propargylamine, in order to avoid the potential toxic effects of free azide groups [57]. Moreover, this final capping step also allows the polymer’s backbone to regain primary amine groups similar to those in unmodified chitosan and to which chitosan owes some of its appealing features, such as its bioadhesive and bacteriostatic properties. 8. Although the CuAAC-based reactions steps can be performed with type 2 water, further washing steps should be undertaken using type 1 water to ensure the removal of any organic impurities which can interfere with characterization techniques or further functionality assays.

Acknowledgments This work was financially supported by Fundac¸˜ao para a Cieˆncia e Tecnologia, Portugal, through projects UIDB/50006/2020 (LAQV-REQUIMTE) and POCI-01-0145-FEDER-031781 (AntINFECT), contract CEECIND/01921/2017/CP1392/ CT0002 to FC, and PhD Grant SFRH/BD/108966/2015 to MB. Thanks are also due to Comissa˜o de Coordenac¸˜ao e Desenvolvimento Regional do Norte (CCDR-N)/NORTE2020/ Portugal2020 for funding through projects DESignBIOtechHealth (ref. Norte-01-0 145-FEDER-000024) and Bioengineered Therapies for Infectious Diseases and Tissue Regeneration (ref. NORTE-01-0145-FEDER-000012).

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16. Fosgerau K, Hoffmann T (2015) Peptide therapeutics: current status and future directions. Drug Discov Today 20(1):122–128 17. McGregor DP (2008) Discovering and improving novel peptide therapeutics. Curr Opin Pharmacol 8(5):616–619 18. Sato AK, Viswanathan M, Kent RB et al (2006) Therapeutic peptides: technological advances driving peptides into development. Curr Opin Biotechnol 17(6):638–642 19. Chandrudu S, Simerska P, Toth I (2013) Chemical methods for peptide and protein production. Molecules 18(4):4373–4388 20. Craik DJ, Fairlie DP, Liras S et al (2013) The future of peptide-based drugs. Chem Biol Drug Des 81(1):136–147 21. Vlieghe P, Lisowski V, Martinez J et al (2010) Synthetic therapeutic peptides: science and market. Drug Discov Today 15(1–2):40–56 22. Kaspar AA, Reichert JM (2013) Future directions for peptide therapeutics development. Drug Discov Today 18(17–18):807–817 23. Coin I, Beyermann M, Bienert M (2007) Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat Protoc 2(12):3247–3256 24. Censi R, Di Martino P, Vermonden T et al (2012) Hydrogels for protein delivery in tissue engineering. J Control Release 161 (2):680–692 25. Vandermeulen GWM, Klok H-A (2004) Peptide/protein hybrid materials: enhanced control of structure and improved performance through conjugation of biological and synthetic polymers. Macromol Biosci 4 (4):383–398 26. Antosova Z, Mackova M, Kral V et al (2009) Therapeutic application of peptides and proteins: parenteral forever? Trends Biotechnol 27(11):628–635 27. Casault S, Kenward M, Slater GW (2007) Combinatorial design of passive drug delivery platforms. Int J Pharm 339(1–2):91–102 28. Furth ME, Atala A, Van Dyke ME (2007) Smart biomaterials design for tissue engineering and regenerative medicine. Biomaterials 28 (34):5068–5073 29. Tessmar JK, Go¨pferich AM (2007) Matrices and scaffolds for protein delivery in tissue engineering. Adv Drug Deliv Rev 59 (4–5):274–291 30. Lalatsa A, Sch€atzlein AG, Mazza M et al (2012) Amphiphilic poly(l-amino acids)—new

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synthesis and potential biomedical applications. Biomacromolecules 8(6):1844–1850 45. Conibear AC, Farbiarz K, Mayer RL et al (2016) Arginine side-chain modification that occurs during copper-catalysed azide–alkyne click reactions resembles an advanced glycation end product. Org Biomol Chem 14 (26):6205–6211 46. Campbell-Verduyn LS, Mirfeizi L, Dierckx RA et al (2009) Phosphoramidite accelerated copper(i)-catalyzed [3 + 2] cycloadditions of azides and alkynes. Chem Commun (16):2139–2141 47. Hong V, Presolski SI, Ma C et al (2009) Analysis and optimization of copper-catalyzed azide–alkyne cycloaddition for bioconjugation. Angew Chem Int Ed 48(52):9879–9883 48. den Hertog AL, Sang HWWF, Kraayenhof R et al (2004) Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization. Biochem J 379(3):665–672 49. Faber C, Hoogendoorn RJW, Stallmann HP et al (2004) In vivo comparison of Dhvar-5 and gentamicin in an MRSA osteomyelitis prevention model. J Antimicrob Chemother 54 (6):1078–1084 50. Ruissen ALA, Groenink J, Van ‘t Hof W et al (2002) Histatin 5 and derivatives: their localization and effects on the ultra-structural level. Peptides 23(8):1391–1399 51. Barbosa M, Costa F, Monteiro C et al (2019) Antimicrobial coatings prepared from Dhvar5-click-grafted chitosan powders. Acta Biomater 84:242–256 52. Castro V, Blanco-Canosa JB, Rodriguez H et al (2013) Imidazole-1-sulfonyl azide-based diazo-transfer reaction for the preparation of azido solid supports for solid-phase synthesis. ACS Comb Sci 15(7):331–334 53. Barbosa M, Vale N, Costa FMTA et al (2017) Tethering antimicrobial peptides onto chitosan: optimization of azide-alkyne “click” reaction conditions. Carbohydr Polym 165:384–393 54. Petrou C, Sarigiannis Y (2018) 1-peptide synthesis: methods, trends, and challenges. In: Koutsopoulos S (ed) Peptide applications in biomedicine, biotechnology and bioengineering. Woodhead Publishing, Sawston, pp 1–21. https://doi.org/10.1016/B978-0-08100736-5.00001-6 55. Kim SE, Harker EC, De Leon AC et al (2015) Coextruded, aligned, and gradient-modified poly(epsilon-caprolactone) fibers as platforms for neural growth. Biomacromolecules 16 (3):860–867

Only a “Click” Away: Development of Peptide-Based Materials 56. Presolski SI, Hong VP, Finn MG (2011) Copper-catalyzed azide–alkyne click chemistry for bioconjugation. Curr Protoc Chem Biol 3 (4):153–162 57. Crownover E, Duvall CL, Convertine A et al (2011) RAFT-synthesized graft copolymers that enhance pH-dependent membrane

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Chapter 5 Fluoroglycoproteins by Copper-Free Strain-Promoted Azide–Alkyne Cycloaddition Pedro M. S. D. Cal, Gonc¸alo J. L. Bernardes, and Omar Boutureira Abstract This chapter describes a reliable two-step, metal-free protocol for the preparation of well-defined fluoroglycoproteins. It starts with a first alkylation step to chemoselectively install strained alkyne handles at cysteine residues followed by a second strain-promoted azide–alkyne cycloaddition using an inverse electron-demand Diels–Alder reaction. This proof-of-principle study that uses the apoptotic protein marker Annexin V enables the efficient metal-free incorporation of 2-deoxy-2-fluoro-glycopyranosyl azides into proteins and complements previous methods using Cu(I)-mediated azide–alkyne cycloadditions and thiol chemistry. Key words Selective protein modification, Bioconjugation, Bioorthogonal reaction, Covalent conjugation chemistry, Homogeneous glycoconjugates, Fluor, Fluorosugars, Fluoroglycoproteins, Strainpromoted azide–alkyne cycloaddition, Inverse electron-demand Diels–Alder reaction

Abbreviations ADIBO AnxV BCN CuAAC DBCO DIFO DMF ESI FDG FDGN3 HPLC LC MS MWCO

Azadibenzobicyclooctyne Annexin V Bicyclo[6.1.0]nonyne Copper-mediated azide–alkyne cycloaddition Dibenzocyclooctyne Difluorinated cyclooctyne N,N-Dimethylformamide Electrospray ionization 2-Deoxy-2-fluoro-β-D-glucopyranose 2-Deoxy-2-fluoro-β-D-glucopyranosyl azide High-performance liquid chromatography Liquid chromatography Mass spectrometry Molecular weight cut-off

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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SPAAC TOF UPLC

1

Strain-promoted azide–alkyne cycloaddition Time of flight Ultra-performance liquid chromatography

Introduction Rational OH-to-F replacements in carbohydrate antigens represent a key strategy for the design of biologically active (glyco)peptides and proteins with improved pharmacological parameters that simultaneously carry a powerful 19F spectroscopic label [1– 3]. Alternatively, the use of 18F provides new opportunities for noninvasive imaging and diagnosis using this positron-emitting isotope [4]. The unique physical and chemical properties of the fluorine atom, such as its small size and high electronegativity, allow the modulation of physical and pharmacological properties of the biomolecule to which is attached (e.g., increased chemical/enzymatic stability, hydrophobicity, etc.) [5]. However, despite the increasing interest in fluorinated pharmaceuticals, including those derived from carbohydrates, the biological consequences of fluorination on the structure and activity of (glyco)peptides and proteins (e.g., how fluorine interacts with protein recognition sites) are still being discussed and rationalized [6, 7]. Recent advances in selective chemical protein modification [8–11] have stimulated the selective incorporation of such synthetic, unnatural F-oligosaccharide mimetics into peptides and proteins mainly using Cu(I)-mediated azide–alkyne cycloadditions [12–16] and thiol chemistry [17–21], which have emerged as versatile and powerful synthetic tools for the preparation of well-defined F-carbohydrate-based vaccines and other detection/diagnosis elements [22–32]. However, avoiding the use of detrimental reaction conditions that may lead to toxicity [33–35] and/or reactivity [36] problems is an important issue still to be addressed. This chapter reports the utilization of a two-step protein-modification protocol for the development of synthetic homogeneous fluorinated glycoproteins via the incorporation of strained alkynes into proteins by either α-haloacetamide or maleimide S-alkylation [37, 38] and subsequent copper-free strain-promoted azide–alkyne cycloaddition (SPAAC) [39, 40] using the apoptotic marker Annexin V (AnxV) and 2-deoxy-2-fluoro-β-D-glucopyranosyl azide (FDGN3) pair as model systems. Studying the reactivity of novel partners and conditions to access fluorinated (glyco)peptides and proteins will provide valuable information to further explore the biological implications of these emerging therapeutics and imaging agents in vivo.

Fglycoproteins by Strain-Promoted Azide–Alkyne Cycloaddition

2

55

Materials All reagents and solvents (analytical or HPLC grade) are used as received from commercial suppliers without prior purification. Milli-QR purified water is used for protein manipulations. Follow all waste disposal regulations when disposing waste materials. 1. 20 mM Tris–HCl buffer, pH 8. 2. N,N-Dimethylformamide (DMF). 3. DBCO-maleimide was purchased from Sigma-Aldrich. 4. DBCO-bromoacetamide was prepared according to Liang et al. [41]. 5. Ellman’s reagent: 14 mg/mL stock solution in H2O. 6. Eppendorf tube: 0.5 mL. 7. Zeba™ Spin desalting column: Thermo Fisher Scientific, 7K MWCO, 0.5 mL. 8. Vivaspin® 500 centrifugal concentrator: Sigma-Aldrich, 5K MWCO, 0.5 mL. 9. Bradford reagent: Sigma-Aldrich. 10. Laboratory centrifuge. 11. Vortex shaker. 12. Eppendorf incubator shaker. 13. MS vials with 300 μL inserts. 14. Standard biological pipettes (various sizes).

2.1 Protein Mass Spectrometry Equipment

Liquid chromatography–mass spectrometry (LC–MS) is performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system (Acquity UPLC BEH300 C4 column, 1.7 μm, 2.1 mm
50 mm) (see Note 1). Water with 0.1% formic acid (solvent A) and 70% acetonitrile and 29.925% water with 0.075% formic acid (solvent B) are used as the mobile phase at a flow rate of 0.2 mL/min. The gradient is programmed as follows: from 72% A to 100% B for 25 min then 100% B for 2 min and 72% A for 18 min. The electrospray source is operated with a capillary voltage of 2.0 kV and a cone voltage of 40 V. Nitrogen is used as the desolvation gas at a total flow of 850 L/h. Total mass spectra are reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v. 4.1 from Waters) according to the manufacturer’s instructions.

2.2 Protein LC–MS/ MS Equipment

All LC–MS/MS experiments are performed using a nanoAcquity UPLC (Waters Corp., Milford, MA) system and an LTQ Orbitrap Velos hybrid ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Separation of peptides is performed by reverse-

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phase chromatography using a Waters reverse-phase nano-column (BEH C18, 1.7 μm, 75 μm
250 mm) at flow rate of 300 nL/min. Peptides are initially loaded onto a pre-column (Waters UPLC Trap Symmetry C18, 5 μm, 180 μm
20 mm) from the nanoAcquity sample manager with 0.1% formic acid for 3 min at a flow rate of 10 μL/min. After this period, the column valve is switched to allow the elution of peptides from the pre-column onto the analytical column. Solvent A is water +0.1% formic acid and solvent B is acetonitrile +0.1% formic acid. The linear gradient employed is 5–40% B in 60 min. The LC eluent is sprayed into the mass spectrometer by means of a New Objective nanospray source. All m/z values of eluting ions are measured in the Orbitrap Velos mass analyzer, set at a resolution of 30000. Data-dependent scans (Top 20) are employed to automatically isolate and generate fragment ions by collision-induced dissociation in the linear ion trap, resulting in the generation of MS/MS spectra. Ions with charge states of 2+ and above are selected for fragmentation. Post-run, the data is processed using Proteome Discoverer (version 1.3., Thermo Fisher Scientific). 2.3 Protein of Interest Containing a Unique Cysteine

3

In this protocol, we use Annexin V (AnxV) that is expressed and purified according to Salvado´ et al. [17]. Sequence of AnxV C315 (modified residue underlined and bold). AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESIL TLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLI VALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELR AIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDP DAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVS HLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSI PAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFR KNFATSLYSMIKGDTSGDYKKALLLLCGEDD. Calculated average isotopic mass ¼ 35805.58 (N-terminal Met cleaved). A typical analysis of a conjugation reaction by LC–MS is described below (Subheading 3.1, Fig. 1). The total ion chromatogram, combined ion series, and deconvoluted spectra are shown for AnxV. Identical analyses are carried out for all the conjugation reactions performed in this chapter.

Methods All manipulations are carried out at room temperature unless otherwise indicated.

3.1 Control Reaction of AnxV with Ellman’s Reagent

1. Transfer a 10 μL aliquot of AnxV (1 mg/mL, 0.616 nmol) in 20 mM Tris–HCl buffer at pH 8 to a 0.5 mL Eppendorf tube.

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Fig. 1 ESI–MS spectrum of AnxV

2. Add Ellman’s reagent (2 μL of a 14 mg/mL stock solution in H2O, 70.65 nmol), and vortex the resulting mixture for 10 s (see Note 2). 3. Incubate the reaction mixture at room temperature (shaking at 500 rpm). 4. Monitor reaction progress by LC–MS until no starting material is detected (see Note 3). After 1 h of additional shaking, a 3 μL aliquot is analyzed by LC–MS (3 μL aliquot diluted by 7 μL of 20 mM Tris–HCl buffer at pH 8), and complete conversion to the expected Ellman’s product (calculated mass, 36003; observed mass, 36003) is observed (Fig. 2). 3.2 Reaction of AnxV with DBCO-Maleimide (S-Alkylation Method 1)

1. In a 0.5 mL Eppendorf tube, dilute 8 μL of a 25 μM solution of AnxV with 28 μL of 20 mM Tris–HCl buffer at pH 8 and 3.5 μL of DMF (see Note 4). 2. Add dibenzocyclooctyne-maleimide (DBCO-maleimide) (see Note 5) (0.5 μL of a 10 mM stock solution in DMF) and vortex the resulting mixture for 10 s (see Note 6). 3. Incubate the reaction mixture at room temperature (shaking at 500 rpm). 4. Monitor reaction progress by LC–MS until no starting material is detected (see Note 3). After 5 days of additional shaking, a 3 μL aliquot is analyzed by LC–MS (3 μL aliquot diluted

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Fig. 2 ESI–MS of the reaction of AnxV with Ellman’s reagent

by 7 μL of 20 mM Tris–HCl buffer at pH 8), and complete conversion to AnxV-S-maleimide-DBCO (calculated mass, 36233; observed mass, 36232) is observed (Fig. 3) (see Note 7). 5. Remove small molecules from the reaction mixture by loading the sample onto a Zeba™ Spin desalting column (Thermo Fisher Scientific, 7K MWCO) previously equilibrated with 20 mM Tris–HCl buffer at pH 8. The sample is eluted by centrifugation (2 min, 1500
g) (see Note 8). 6. Concentrate the protein sample to 10 μM (by Bradford assay) [42] using a Vivaspin® 500 centrifugal concentrator (SigmaAldrich, 5K MWCO). 7. Flash frozen the sample with liquid nitrogen and store at 20  C. 3.3 LC–MS/MS Analysis

1. A 10 μM solution of the purified AnxV-S-maleimide-DBCO is enzymatically digested by trypsin overnight and subjected to LC–MS/MS analysis according to Cal et al. [43]. No reduction/alkylation steps are performed. 2. Briefly, all MS/MS data is converted to mgf files, and these are submitted to the Mascot search algorithm (Matrix Science, London, UK) and searched against a custom database

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Fig. 3 ESI–MS of the reaction of AnxV with DBCO-maleimide

containing the corresponding protein and applying variable modifications of oxidation (M), deamination (NQ), and a custom modification (C), using a peptide tolerance of 25 ppm (MS) and 0.8 Da (MS/MS). Peptide identifications are accepted if they could be established at greater than 95.0% probability. 3. Significant hits, which suggested that the maleimide-DBCO modification is bound to peptides, are then verified by manual inspection of the MS/MS data. Mascot search shows that ALLLLCGEDD is detected with the expected modification at cysteine. The MS/MS spectrum is depicted in the following figure with the majority of the sequence ions assigned (Fig. 4) (see Note 9).

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Fig. 4 MS/MS spectrum of the m/z 744.84 (2+) ion of the tryptic peptide ALLLLCGEDD from AnxV-Smaleimide-DBCO, containing the maleimide-DBCO modification at the original cysteine residue. The generated fragment ions are consistent with the mass of the modification

3.4 Reaction of AnxV with DBCOBromoacetamide (S-Alkylation Method 2)

1. In a 0.5 mL Eppendorf tube, dilute 8 μL of a 25 μM solution of AnxV with 28 μL of 20 mM Tris–HCl buffer at pH 8. 2. Add dibenzocyclooctyne-α-bromoacetamide (DBCObromoacetamide) (4 μL of a 10 mM stock solution in DMF), and vortex the resulting mixture for 10 seconds (see Note 6). 3. Incubate the reaction mixture at room temperature (shaking at 500 rpm). 4. Monitor reaction progress by LC–MS until no starting material is detected (see Note 3). After 5 days of additional shaking, a 3 μL aliquot is analyzed by LC–MS (3 μL aliquot diluted by 7 μL of 20 mM Tris–HCl buffer at pH 8), and complete conversion to AnxV-S-acetamide-DBCO (calculated mass, 36122; observed mass, 36121) is observed (Fig. 5) (see Note 7). 5. Remove small molecules from the reaction mixture by loading the sample onto a Zeba™ Spin desalting column (Thermo Fisher Scientific, 7K MWCO) previously equilibrated with 20 mM Tris–HCl buffer at pH 8. The sample is eluted by centrifugation (2 min, 1500
g) (see Note 8).

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Fig. 5 ESI–MS of the reaction of AnxV with DBCO-bromoacetamide

6. Concentrate the protein sample to 10 μM (by Bradford assay) [42] using a Vivaspin® 500 centrifugal concentrator (SigmaAldrich, 5K MWCO). 7. Flash frozen the sample with liquid nitrogen and store at 20  C. 3.5 Reaction of AnxV-S-MaleimideDBCO with 2-Deoxy-2fluoro-β-Dglucopyranosyl Azide

1. Prepare AnxV-S-maleimide-DBCO as a 5 μM solution in 20 mM Tris–HCl buffer at pH 8 and transfer 30 μL to a 0.5 mL Eppendorf tube. 2. Add 2-deoxy-2-fluoro-β-D-glucopyranosyl azide (FDGN3) (0.4 μL of a 48.3 mM stock solution in DMF) and vortex the resulting mixture for 10 s (see Note 10). 3. Incubate the reaction mixture at room temperature (shaking at 500 rpm). 4. Monitor reaction progress by LC–MS until no starting material is detected (see Note 3). After 19 h of additional shaking, a 3 μL aliquot is analyzed by LC–MS (3 μL aliquot diluted by 7 μL of

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Fig. 6 ESI–MS of the reaction of AnxV-S-maleimide-DBCO with 2-deoxy-2-fluoro-β-D-glucopyranosyl azide (FDGN3)

20 mM Tris–HCl buffer at pH 8), and complete conversion to AnxV-S-maleimide-DBCO-FDG (calculated mass, 36439; observed mass, 36440) is observed (Fig. 6) (see Note 11). 5. Remove small molecules from the reaction mixture by loading the sample onto a Zeba™ Spin desalting column (Thermo Fisher Scientific, 7K MWCO) previously equilibrated with 20 mM Tris–HCl buffer at pH 8. The sample is eluted by centrifugation (2 min, 1500
g). 6. Flash frozen the sample with liquid nitrogen and store at 20  C (see Note 12). 7. The hydrolysis of the maleimide conjugate is evaluated upon incubation at room temperature for 2 weeks. After this period, ca. 30% maleimide hydrolysis (calculated mass, 36457; observed mass, 36457) and ca. 30% retro-Michael to AnxV (calculated mass, 35806; observed mass, 35805) is observed (Fig. 7).

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Fig. 7 ESI–MS of AnxV-S-maleimide-DBCO-FDG upon incubation at room temperature for 2 weeks

3.6 Reaction of AnxV-S-AcetamideDBCO with 2-Deoxy-2fluoro-β-Dglucopyranosyl Azide

1. Prepare AnxV-S-acetamide-DBCO as a 5 μM solution in 20 mM Tris–HCl buffer at pH 8 and transfer 20 μL to a 0.5 mL Eppendorf tube. 2. Add 2-deoxy-2-fluoro-β-D-glucopyranosyl azide (FDGN3) (2.1 μL of a 48.3 mM stock solution in DMF), and vortex the resulting mixture for 10 s (see Note 10). 3. Incubate the reaction mixture at room temperature (shaking at 500 rpm). 4. Monitor reaction progress by LC–MS until no starting material is detected (see Note 3). After 14.5 h of additional shaking, a 3 μL aliquot is analyzed by LC–MS (3 μL aliquot diluted by 7 μL of 20 mM Tris–HCl buffer at pH 8), and complete conversion to AnxV-S-acetamide-DBCO-FDG (calculated mass, 36329; observed mass, 36329) is observed (Fig. 8) (see Note 11). 5. Remove small molecules from the reaction mixture by loading the sample onto a Zeba™ Spin desalting column (Thermo Fisher Scientific, 7K MWCO) previously equilibrated with 20 mM Tris–HCl buffer at pH 8. The sample is eluted by centrifugation (2 min, 1500
g). 6. Flash frozen the sample with liquid nitrogen and store at 20  C (see Note 12).

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Fig. 8 ESI–MS of the reaction of AnxV-S-acetamide-DBCO with 2-deoxy-2-fluoro-β-D-glucopyranosyl azide (FDGN3)

4

Notes 1. The use of liquid chromatography–mass spectrometry (LC– MS) analysis is recommended for monitoring the reaction progress on peptides and proteins with accurate resolution. 2. The use of chemical controls such as the cysteine-specific Ellman’s reagent allows for determining not only the presence of free cysteine residues but also their relative reactivity under the conditions tested, taking into consideration their native residue microenvironment. 3. Reaction monitoring by LC–MS analysis determines the minimum reaction time necessary to achieve full conversion. Extended reaction times are typically associated to a decrease of final protein concentration.

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4. Same reaction was attempted on a larger scale (
10), but the results were not reproducible, and extra 13 equiv of maleimideDBCO was required to achieve full conversion after 11 days at room temperature. Importantly, the amount of organic solvent used should be adjusted to up to 10% to avoid protein precipitation and/or denaturing. 5. A number of structurally varied cyclooctynes (e.g., DIFO, BCN, and ADIBO, among others) have been developed featuring different reaction kinetics and hydrophobicity/philicity [39, 40]. Among them, dibenzocyclooctyne (DBCO)-based reagents combine high reactivity and sufficient hydrophilicity, thus reducing non-specific binding while having greater efficiency than common copper-mediated azide–alkyne cycloadditions (CuAAC). 6. We freshly prepare this solution prior to each experiment. 7. Optimization of assay conditions described above is required (protein concentration/reaction scale, buffer composition and pH, amount of alkylating agent, % of organic co-solvent, and reaction time and temperature) [43]. 8. This desalting step is essential to remove excess alkyne reagent that will react with FDGN3 in the next step. 9. Proteolytic digest followed by peptide mapping by LC–MS/ MS should confirm the desired site of modification and the absence of additional modifications at other residues. 10. The FDGN3 solution can be stored at 4  C for several months. 11. No catalyst or accessory reagents and thus no extensive optimization of assay conditions required [44] unlike for CuAAC reactions where optimization is typically required (type and concentration of copper source, reduction reagent, ligands, and co-solvents) [45]. 12. Following the two consecutive reactions described above, proteins should be observed by LC–MS as a single species with >95% conversion to the desired final product, as judged by calculation from peak intensities in the deconvoluted spectrum.

Acknowledgments The authors thank the Spanish Government (MCIU) (CTQ201790088-R to O.B.), the national agency of investigation (AEI), the European Regional Development Fund, the Royal Society (URF to G.J.L.B., URF/R/180019), FCT Portugal (iFCT to G.J.L.B., IF/00624/2015 and postdoctoral fellowship to P.M.S.D.C., SFRH/BPD/103172/2014), and the EPSRC (EP/M003647/1 to G.J.L.B.) for financial support. O.B. is a Ramo´n y Cajal Fellow (RYC-2015-17705).

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References ˜ o B, Postigo A (2019) Syn1. Uhrig ML, Lantan thetic strategies for fluorination of carbohydrates. Org Biomol Chem 17:5173–5189 2. Gillis EP, Eastman KJ, Hill MD, Donnelly DJ, Meanwell NA (2015) Applications of fluorine in medicinal chemistry. J Med Chem 58:8315–8359 3. Purser S, Moore PR, Swallow S, Gouverneur V (2008) Fluorine in medicinal chemistry. Chem Soc Rev 37:320–330 4. Maschauer S, Prante O (2014) Sweetening pharmaceutical radiochemistry by 18F-fluoroglycosylation: a short review. Biomed Res Int 2014:1–16 5. Hunter L (2010) The C–F bond as a conformational tool in organic and biological chemistry. Beilstein J Org Chem 6:38 6. Arda´ A, Jime´nez-Barbero J (2018) The recognition of glycans by protein receptors. Insights from NMR spectroscopy. Chem Commun 54:4761–4769 7. Arntson KE, Pomerantz WCK (2015) Proteinobserved fluorine NMR: a bioorthogonal approach for small molecule discovery. J Med Chem 59:5158–5171 8. Li C, Wang LX (2018) Chemoenzymatic methods for the synthesis of glycoproteins. Chem Rev 118:8359–8413 9. Boutureira O, Bernardes GJL (2015) Advances in chemical protein modification. Chem Rev 115:2174–2195 10. Krall N, da Cruz FP, Boutureira O, Bernardes GJL (2015) Site-selective protein-modification chemistry for basic biology and drug development. Nat Chem 8:103–113 11. Spicer CD, Davis BG (2014) Selective chemical protein modification. Nat Commun 5:4740 12. Boutureira O, D’Hooge F, Ferna´ndezGonza´lez M, Bernardes GJL, Sa´nchezNavarro M, Koeppe JR, Davis BG (2010) Fluoroglycoproteins: ready chemical siteselective incorporation of fluorosugars into proteins. Chem Commun 46:8142–8144 13. Ferna´ndez-Gonza´lez M, Boutureira O, Bernardes GJL, Chalker JM, Young MA, Errey JC, Davis BG (2010) Site-selective chemoenzymatic construction of synthetic glycoproteins using endoglycosidases. Chem Sci 1:709–715 14. Vala C, Chre´tien F, Balentova E, Lamande´-Langle S, Chapleur Y (2011) Neoglycopeptides through direct functionalization of cysteine. Tetrahedron Lett 52:17–20

15. Maschauer S, Prante O (2009) A series of 2-Otrifluoromethylsulfonyl-D-mannopyranosides as precursors for concomitant 18F-labeling and glycosylation by click chemistry. Carbohydr Res 344:753–761 16. Maschauer S, Einsiedel J, Haubner R, Hocke C, Ocker M, Hu¨bner H et al (2010) Labeling and glycosylation of peptides using click chemistry: a general approach to 18F-glycopeptides as effective imaging probes for positron emission tomography. Angew Chem Int Ed 49:976–979 17. Salvado´ M, Amgarten B, Castillo´n S, Bernardes GJL, Boutureira O (2015) Synthesis of fluorosugar reagents for the construction of welldefined fluoroglycoproteins. Org Lett 17:2836–2839 18. Boutureira O, Bernardes GJL, Ferna´ndezGonza´lez M, Anthony DC, Davis BG (2012) Selenenylsulfide-linked homogeneous glycopeptides and glycoproteins: synthesis of human “hepatic Se metabolite A”. Angew Chem Int Ed 51:1432–1436 19. Boutureira O, Bernardes GJL, D’Hooge F, Davis BG (2011) Direct radiolabelling of proteins at cysteine using [18F]-fluorosugars. Chem Commun 47:10010–10012 20. Fro¨hlich RFG, Schrank E, Zangger K (2012) 2,2,2-Trifluoroethyl 6-thio-β-D-glucopyranoside as a selective tag for cysteines in proteins. Carbohydr Res 361:100–104 21. Prante O, Einsiedel J, Haubner R, Gmeiner P, Wester H-J, Kuwert T et al (2007) 3,4,6-TriO-acetyl-2-deoxy-2-[18F]fluoroglucopyranosyl phenylthiosulfonate: a thiol-reactive agent for the chemoselective 18F-glycosylation of peptides. Bioconjug Chem 18:254–262 22. Collet C, Maskali F, Cle´ment A, Chre´tien F, Poussier S, Karcher G et al (2015) Development of 6-[18F]fluoro-carbohydrate-based prosthetic groups and their conjugation to peptides via click chemistry. J Label Compd Radiopharm 59:54–62 23. Fischer CR, Mu¨ller C, Reber J, Mu¨ller A, Kr€amer SD, Ametamey SM et al (2012) [18F] Fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting. Bioconjug Chem 23:805–813 24. Wuest F, Berndt M, Bergmann R, van den Hoff J, Pietzsch J (2008) Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective

Fglycoproteins by Strain-Promoted Azide–Alkyne Cycloaddition 18 F-labeling of peptides and proteins. Bioconjug Chem 19:1202–1210 25. Namavari M, Cheng Z, Zhang R, De A, Levi J, Hoerner JK et al (2009) A novel method for direct site-specific radiolabeling of peptides using [18F]FDG. Bioconjug Chem 20:432–436 26. Hultsch C, Schottelius M, Auernheimer J, Alke A, Wester H-J (2009) 18F-Fluoroglucosylation of peptides, exemplified on cyclo (RGDfK). Eur J Nucl Med Mol Imaging 36:1469–1474 27. Yang F, Zheng X-J, Huo C-X, Wang Y, Zhang Y, Ye X-S (2011) Enhancement of the immunogenicity of synthetic carbohydrate vaccines by chemical modifications of STn antigen. ACS Chem Biol 6:252–259 28. Huo C-X, Zheng X-J, Xiao A, Liu C-C, Sun S, Lv Z et al (2015) Synthetic and immunological studies of N-acyl modified S-linked STn derivatives as anticancer vaccine candidates. Org Biomol Chem 13:3677–3690 29. Lee H-Y, Chen C-Y, Tsai T-I, Li S-T, Lin K-H, Cheng Y-Y et al (2014) Immunogenicity study of globo H analogues with modification at the reducing or nonreducing end of the tumor antigen. J Am Chem Soc 136:16844–16853 30. Orwenyo J, Huang W, Wang L-X (2013) Chemoenzymatic synthesis and lectin recognition of a selectively fluorinated glycoprotein. Bioorg Med Chem 21:4768–4777 31. Oberbillig T, Mersch C, Wagner S, HoffmannRo¨der A (2012) Antibody recognition of fluorinated MUC1 glycopeptide antigens. Chem Commun 48:1487–1489 32. Hoffmann-Ro¨der A, Kaiser A, Wagner S, Gaidzik N, Kowalczyk D, Westerlind U et al (2010) Synthetic antitumor vaccines from tetanus toxoid conjugates of MUC1 glycopeptides with the Thomsen-Friedenreich antigen and a fluorine-substituted analogue. Angew Chem Int Ed 49:8498–8503 33. Hong V, Presolski SI, Ma C, Finn MG (2009) Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew Chem Int Ed 48:9879–9883 34. Besanceney-Webler C, Jiang H, Zheng T, Feng L, Soriano del Amo D, Wang W et al (2011) Increasing the efficacy of bioorthogonal click reactions for bioconjugation: a comparative study. Angew Chem Int Ed 50:8051–8056 35. Kennedy DC, McKay CS, Legault MCB, Danielson DC, Blake JA, Pegoraro AF et al

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Chapter 6 Hybrid Silylated Peptides for the Design of Bio-functionalized Materials Titouan Montheil, Ce´cile Echalier, Jean Martinez, Ahmad Mehdi, and Gilles Subra Abstract Hybrid silylated peptides are useful starting compounds for the design of functionalized materials. Indeed, silylated peptides may react in soft conditions with other silylated species through Si–O–Si bonds, opening the way to design novel peptide-based materials and architectures. Herein, we present the various methods allowing the design of bio-functionalized materials with hybrid silylated peptides. These hybrid peptides could either be used as they are (bottom-up synthesis) or to functionalize materials (grafting to). Key words Peptide, Sol-gel, Material, Functionalization, Siloxane

1

Introduction Hybrid silylated peptides comprise a bioactive sequence and at least a hydroxysilane group. This type of building block is of high interest to functionalize materials and to design novel hybrid bioorganic-inorganic structures by the sol-gel process. Hydrolysis of alkoxysilanes and chlorosilanes (i.e., Si–OR or Si–Cl, respectively) into hydroxysilanes (Si–OH) and concomitant condensation yields to siloxane (Si–O–Si) network of covalent bonds, in chemoselective conditions compatible with all amino acid side chains. Hydroxysilanes may react with other metal oxides in a chemoselective way, yielding Si–O–metal covalent bonds, which is interesting for device surface functionalization. Silica, glass, silicon, and silicone surfaces can be modified straightforwardly using this strategy. Besides, silylated peptides may react in soft conditions with other silylated species (e.g., drugs, biopolymers, probes, assembling) through Si–O–Si bonds, opening the way to design novel peptide-based materials and architectures. The first challenge for the biomolecular chemist is to introduce the silyl group at a desired position in the peptide sequence. Then, this hybrid peptide is isolated before being

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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involved in the preparation and the forming of the biomaterial by the sol-gel process.

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Materials All reagents and solvents were purchased from Alfa Aesar, Acros, Sigma-Aldrich, or Merck and were used without further purification.

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Hybrid Peptides

3.1 Reacting Hybrid Peptides by the Sol-Gel Process

Hybrid peptides are elementary building blocks used for the functionalization and the direct synthesis of materials by the sol-gel process. The sol-gel process refers to an inorganic polymerization of molecular precursors to form a metal oxide network. In fact, precursors are generally metal alkoxides of M(OR)n general formula in which M is a metal (Si, Al, Ti, etc.) and R an alkyl group (Me, Et, iPr, etc.). Alternatively, metal halides can be used (MXn, with X ¼ Cl generally). The sol-gel process is characterized by a sol to gel transition. The sol is a stable suspension of colloidal particles within a liquid, and a gel is a three-dimensional interconnected solid network holding large amounts of solvent. Interestingly, this process is compatible with a lot of processing methods, e.g. spin and dip coating, that lead to a wide range of products including fibers, nanoparticles, coatings, thin films, xerogels, aerogels, etc. [1]. It enables the preparation of advanced tailor-made materials. The main advantage of sol-gel for the design of peptide materials is the orthogonality of the reaction. Indeed, since the high stability of Si–O–Si bonds (by comparison to Si–NR, Si–OR, for instance), the hydroxysilanes do not react with most of peptide side chains (including primary amines, alcohols, and carboxylic acids), but more selectively with M–OH groups, leaving the bioactive peptide unmodified. An additional attractive feature is the mild conditions in which sol-gel process can be performed. Indeed, the reaction occurs in water, at room temperature, and physiological pH, which are essential for the synthesis of biomaterials. The sol-gel process is based on two main steps, hydrolysis and condensation, leading to the formation of Si–O–Si bonds. The process starts with the hydrolysis of alkoxysilyl (Si–OR) and chlorosilyl (Si–Cl) groups into silanols (Si–OH) with the resulting release of an alcohol (ROH) and hydrochloric acid (HCl), respectively (Fig. 1a). Then, the condensation step leads to the formation of siloxane bonds (Si–O–Si). When the condensation reaction occurs between two hydroxysilyl groups with release of water, it is called oxolation (Fig. 1b). When it involves a hydroxide and an alkoxide with release of an alcohol, it is called alkoxolation

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Fig. 1 (a) Hydrolysis of a silicon alkoxide/chloride into silanol; (b) condensation via oxolation; (c) condensation via alkoxolation

(Fig. 1c). At neutral pH, these reactions are very slow [2]. However, acids, bases, and nucleophiles can be used as catalysts to increase the rate of these reactions. Sol-gel reactions involving hybrid peptide result in different types of covalent networks depending on the types of silane precursors (e.g., dimethylchloro-, methyldichloro-, triethoxy-silanes). Each methyl group on the silicon atom reduces one possibility of connection with another silane moiety, yielding either a 3D network or a single dimerization in the case of dimethyl hydroxysilane cross-reactions. 3.2 Introduction of a Silyl Group 3.2.1 Silyl Groups

Many types of silylated reagents are commercially available to modify peptides, either as alkoxysilane or chlorosilane derivatives. During the sol-gel process, these functions are hydrolyzed into Si–OH moieties. One to three hydroxyl groups can be present on the same silicon atom, depending on the number of methyl groups displayed on the same atom. After condensation, it results in networks with different connectivities (Fig. 2). From a nomenclature point of view, the non-condensate state is noted M0 (e.g., –SiOHMe2), D0 (e.g., –Si(OH)2Me), or T0 (e.g., –Si(OH)3); “0” mentioning means that silicon is not condensed at all. The totally condensed state of silicon is M1, D2, or T3 (Fig. 2). It is worth noting that partially condensed states can be observed by 29Si NMR spectroscopy. For example, T-species of silicon bearing one to three Si–O–Si linkages are noted T1, T2, or T3, respectively (see Subheading 3.2.3). Typically, alkoxysilanes are more stable than chlorosilanes. Indeed, chlorosilanes are easily hydrolyzed in corresponding silanols, resulting in a difficult isolation of chlorosilane-containing peptides. Nonetheless, when –SiR2Cl are used, silylated

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Fig. 2 Different connectivities in silicon derivatives

compounds can be easily recovered as dimers (see Subheading 4.1). Alkoxysilanes are more stable in water, and a catalyst is generally needed for their hydrolysis. Note that the hydrolysis rate is depending on the nature of the alkoxy group (i.e., hydrolysis rate, OMe > OEt > OiPr). 3.2.2 Introduction of the Silane Function on the Peptide (i.e., Peptide Silylation)

Many types of reactions can be envisioned to react a silylated reagent to a peptide. However, chemistries to form amides and ureas using commercially available reagents are among the most popular. Activated carboxylic acids on peptides will react with amine-containing silane reagents such as aminopropyltriethoxysilane (APTES) [3]. On the other hand, isocyanate reagents are also very popular since they are available with various silyl groups (–Si (Me)2Cl, –SiMe(Cl)2, –Si(OMe)3, –Si(OEt)3, etc.) and react easily with free amino groups present on the peptide (Fig. 3).

3.2.3 Analysis

Monitoring the silylation of the peptide can be performed by LC/MS. Chlorosilanes could never be detected as they are hydrolyzed during the preparation of the sample. Although ethoxysilanes are slowly hydrolyzed, they are barely observed, except if they are injected immediately after the dissolution and when short gradients

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Fig. 3 Peptide silylation by reaction between amino group and isocyanate

( 12.5). Protocol 5 [7]: Example of a Dimer Synthesis (Fig. 10)

1. Prepare the hybrid peptide as described previously (e.g., protocol 1) using isocyanopropyl dimethylchlorosilane as silylating reagent.

Fig. 10 Dimerization at the N-terminus, C-terminus, or at the side chain between two silylated peptides

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Fig. 11 Silicon-based peptide polymers from bis-silylated peptides

2. The hybrid peptide can be purified if necessary on Agilent PLRP-S column. 3. The hybrid peptide is dissolved in phosphate buffer (DPBS pH 7.4, 1) and freeze-dried to recover the dimer. 4.2 Silicone-Based Peptide Polymers (2D) 4.2.1 Bis-Silylated Peptide

When two dimethylchlorosilane functions are introduced on the same peptide sequence (e.g., at the C- and N-termini), the resulting bifunctional hybrid blocks may polymerize. Interestingly, the connecting bond between peptide sequences is a bis-dimethylsiloxane, similar to the silicone polydimethylsiloxane chains (Fig. 11). Such hybrid polymers are refereed as silicone-based peptide polymers [11]. Bifunctional dimethylchlorosilane hybrid peptides are prepared following the same strategies as monofunctional ones, followed by selective removal of the protecting groups of two sites of the peptide (e.g., N-terminus and modified C-terminus, or N-α and N-ε of a N-terminus lysine; Fig. 11, top and bottom, respectively), and then silylation. The polymerization proceeds and leads to hybrid polymers with a molecular weight (Mn) from 7000 to 24,000 g/mol, according to the type of the silylated peptide that is used [11]. Protocol 6 [11]: Example of Synthesis of a Silicon-Based Peptide Polymer from a Bis-Silylated Peptide (Fig. 11)

1. Dissolve the bis-dimethylsiloxane hybrid peptide in a TFA/water solution (1/1000 v/v) at 0.5 g/mL (~0.5 mM). 2. Add phosphate buffer (DPBS pH 7.4, 1) dropwise under stirring to slowly increase the pH of the reaction mixture above 7. Condensation takes place during buffer addition and induces polymer precipitation. 3. Freeze-dry the medium to recover the polymer as a white powder or a sticky gel depending on the peptide sequence.

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Fig. 12 Silicon-based peptide polymers from dihydroxymethyl silylated peptides 4.2.2 Monosilylated Peptides

Another strategy to prepare comb-like polymers is to use monosilylated peptides bearing a single dichloromethylsilane function [13]. Polymerization yields a siloxane backbone very similar to polydimethylsiloxane (PDMS), except that one out of two methyl groups is replaced by pendant peptide chains. Relatively short oligomers are obtained (i.e., 10,000 g/mol). The protocol is similar to protocol 6. Moreover, methyldihydroxysilane-modified peptides can also be copolymerized with dimethyldichlorosilane (Me2SiCl2) to get peptide-functionalized silicone oils with a PDMS-like backbone [14]. Protocol 7 [14]: Example of Synthesis of a Peptide-Modified Silicone (Fig. 12)

1. Dissolve dimethyldichlorosilane in water, and add sodium dodecyl sulphate ([SDS] ¼ 16.4 mM) as a surfactant. 2. Add the silylated peptide (0.01–0.11 mol%), and heat at 60  C for 24 h. 3. Add phosphate buffer (DPBS pH 7.4, 1) dropwise under stirring to slowly increase the pH of the reaction mixture above 7. Condensation takes place during buffer addition and induces polymer precipitation. 4.3 Hybrid PeptideContaining Matrices (3D)

When trialkoxysilane (e.g., Si(OR)3) derivatives are used, the sol-gel inorganic polymerization may occur in the three dimensions, a silicon being connected with up to three other silicone atoms through Si–O–Si bonds. Interestingly, such hybrid trialkoxysilylated blocks can be mixed with other silane derivatives, to get functional bioorganic-inorganic hybrid materials.

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Fig. 13 Direct synthesis of an OMS functionalized with a hybrid peptide 4.3.1 Peptide-Silica Hybrid Matrices

A hybrid bioorganic ordered mesoporous silica (OMS) was obtained in one step by combining hybrid protected peptides (i.e., Boc-ProProAsp(OtBu)Lys[CO-(CH2)3Si(OEt)3]-NH2) with tetraethyl orthosilicate (TEOS) and pluronic P123 (PEO20POP70PEO20) as a structure-directing agent (protocol 8) [15]. The peptide sequence, H-ProProAsp, was described to catalyze aldolization in solution, and the resulting hybrid materials prove to be supported catalysts demonstrating that peptides kept their properties. Protocol 8 [15]: Example of Hybrid Bioorganic Ordered Mesoporous Silica Synthesis Functionalized with a Hybrid Peptide (Fig. 13)

1. Dissolve P123 (0.21 equiv) in a solution of hydrochloric acid (pH 1.5) at 40 mL/g, under stirring for 20 min till dissolution. 2. Add this solution to a mixture containing the TEOS-hybrid peptide (99.8/0.2 mol/mol) (1 equiv). 3. Stir vigorously for 2 h at room temperature until obtaining a homogeneous and clear solution. 4. Heat the solution at 60  C and add NaF (0.4 equiv) to induce polymerization process. 5. Stir for 3 days, filter, and wash five times copiously with ethanol. 6. Remove the surfactant by Soxhlet extraction with ethanol for 24 h. 7. Add 5 mL of TFA/methylene chloride (2/1 v/v) and stir for 1 h, filter, and wash with 10 mL of DMF-DIEA (0.5%), DMF, DCM, and diethyl ether (three times each) to remove peptide-protecting groups. 8. Dry under vacuum.

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Fig. 14 Synthesis of a hybrid PEG (up) or a peptide (down) hydrogels

4.3.2 Hybrid Hydrogels

As sol-gel reaction may proceed in water in mild conditions, it is a valuable strategy to prepare chemical cross-linked hydrogel networks for biomedical and cell-culture applications. By contrast to physical hydrogels whose structure is maintained by weak interactions (e.g., alginate hydrogels), hybrid hydrogels display better mechanical properties and higher stability, thanks to covalent bonds between elements of the network. Hybrid PEG-based hydrogels containing bioactive peptides (i.e., displaying antibacterial properties or promoting cell adhesion) were prepared by mixing hybrid peptides with bis-silylated PEG2000 to yield hydrogels (90% water) in a one-pot procedure (Fig. 14, up) [16]. Noteworthy, the sol-gel reaction was catalyzed by fluorine ions at a concentration allowing cell growth [17].

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More interestingly, a collagen-biomimetic covalent hydrogel was prepared using only hybrid bis-silylated peptides with no PEG (Fig. 14, down). A low-molecular-weight Ac-Lys(ProHypGly)3Lys-NH2 undecapeptide inspired from the collagen consensus sequence was prepared on solid support and silylated on both lysine side chains in solution using ICPTES. The bis-silylated block was polymerized in a “biological buffer” containing mesenchymal stem cells, demonstrating its potency as a biomimetic scaffold [18]. Protocol 9 [16]: Example of Synthesis of a Hybrid PEG Hydrogel Functionalized with a Hybrid Peptide

1. Dissolve the hybrid PEG (10 wt%) in phosphate buffer (DPBS pH 7.4, 1). 2. Add the hybrid peptide to this solution at a concentration of 1 wt% (4.0 mM). 3. Add NaF (0.3 wt%) as catalyst. 4. Place the hybrid solution at 37  C overnight to obtain the PEG/peptide hydrogel. Protocol 10 [18]: Example of Synthesis of a Hybrid Peptide Hydrogel

1. Dissolve the hybrid peptide (10 wt%) in phosphate buffer (DPBS pH 7.4, 1). 2. Add NaF (0.3 wt%) as catalyst. 3. Place the hybrid solution at 37  C overnight to obtain the peptide hydrogel. 4.3.3 Nanostructured Bioorganic-Inorganic Materials

These materials are similar to Subheading 3.1 but no silica (i.e., TEOS) is added.

Templated Assembly of Nanostructured Bioorganic-Inorganic Materials

Maggini et al. have reported degradable hybrid organosilica particles (nanodonuts) composed of a single hybrid block: a short peptide sequence (tri-L-lysine) modified by ICPTES (protocol 11) [19]. Protocol 11 [19]: Example of Templated Assembly of a Hybrid Material (Fig. 15)

1. Dissolve the hybrid peptide in DMF. 2. Add this solution dropwise to a basic aqueous solution of cetyltrimethylammonium bromide (CTAB). 3. Vigorous stirring at 50  C for 2 h. 4. Centrifuge and wash the recovered material with distilled water. 5. Remove the CTAB by Soxhlet extraction with ethanol for 24 h. 6. Dry under vacuum.

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Fig. 15 Synthesis of silylated lysine nanodonuts

Fig. 16 Synthesis of a structured hybrid material Self-Assembly of Nanostructured Bioorganic-Inorganic Materials

Peptides with self-recognition properties may self-assemble into organized structures. They are generally obtained by self-assembly through weak interactions like hydrogen bonding, π–π interactions, etc. [20, 21]. Perhaps one of the most famous self-assembling peptide sequences is the PhePhe dipeptide, and its derivatives are well-known to self-assemble in solution thanks to π–π interactions and other non-covalent bonds [22]. The presence of silane groups on self-assembling peptides may be used to “freeze” covalently the organized structure by inorganic polymerization. We recently reported the first example of nanostructured materials obtained via the so-called “self-mineralization” approach. The PhegemPhe pseudodipeptide sequence was silylated at both amino extremities with ICPTES. Self-assembly of the building block was performed in water, and a lamellar material was obtained [23]. Protocol 12 [23]: Example of Synthesis of a Structured Hybrid Material (Fig. 16)

1. Put in suspension the hybrid peptide (38.5 mM) in a solution of hydrochloric acid (pH 1.5). 2. Stirring of the heterogeneous mixture for 4 days.

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3. Filter the white solid that precipitated. 4. Wash with a solution of hydrochloric acid (pH 1.5), and dry under vacuum for 40 min at 50  C.

5

Surface Functionalization with Hybrid Peptides Silylated peptides are attractive one-in-all reagents to covalently functionalize surfaces through Si–O–M bonds, M being a metal (e.g., Si, Ti, Al, Sn, etc.) and preferentially a silicon atom. In the latter case, silicon, silica, glass, and even silicone surfaces can be grafted with the desired peptide in one step. In some cases, the surface of the material has to be activated to generate functions (e.g., Si–OH) suitable for anchoring.

5.1 Grafting on Silicones

Silicone and PDMS are extensively used for medical devices, mainly because they are biocompatible synthetic polymers [24]. Nonetheless, such materials are bioinert and have to be functionalized when specific properties are required. Because there is no reactive function on the surface, the material has to be activated [14]. Oxygen plasma treatment generates Si–OH functions suitable for hybrid peptide’s anchoring. Dimethylchlorosilane peptide was thus reacted with Si–OH functions. Yielding a single Si–O–Si bond on the surface, it guarantees the orientation of the peptide and the formation of a single layer of biomolecule on the PDMS. Two examples were reported so far. Peptides derived from extracellular matrix proteins were selected to promote wound healing once grafted on commercial silicone dressings. Catheters grafted with antibacterial peptide showed better efficiency in the short term (first 7 days after contamination) than silver-doped commercial devices [25]. Whatever the device, the dip coating procedure was used after oxygen plasma activation of the material. Protocol 13 [25]: Example of Silicone Functionalization with a Hybrid Peptide (Fig. 17)

1. Dissolve the hybrid peptide (10 mM) in water (1/1000 TFA, v/v)/ethanol, (15/85, v/v). 2. Submit silicone dressings to 90 s oxygen plasma treatment with a low-pressure chamber plasma apparatus (Diener Electronics; settings 13.56 MHz radiofrequency, 0.6 mbar, 60 W). 3. Immediately after plasma activation, dip the silicone pieces into the hybrid peptide solution (velocity 5 cm/min). 4. Place coated silicone pieces at 50  C for 24 h for aging. 5. Wash with a water/ethanol solution (3/7 v/v) for 5 min.

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Fig. 17 Silicone functionalization with hybrid peptides 5.2 Grafting of Peptides on Porous Silica, Glass, and Titanium Surfaces 5.2.1 Silica Grafting

Silica grafting is perhaps the most straightforward grafting method, as silica displays numerous Si–OH groups on the surface. Nonfunctionalized OMS nanoparticles were grafted with Boc-ProProAsp(OtBu)Lys[CO-(CH2)3Si(OEt)3]-NH2 [3]. This mesoporous bioorganic-inorganic peptide hybrid silica was used as an aldolization catalyst and was found to be as efficient as the OMS obtained in a single step by direct synthesis (see Subheading 3.1). Protocol 14 [3]: Example of OMS Functionalization with a Hybrid Peptide

1. Dissolve the hybrid peptide in DMF. 2. Add OMS particles in suspension (1/10 w/w hybrid peptideOMS), and stir for 1 h at room temperature and then for 24 h at 80  C. 3. Filter the OMS hybrid, wash with ethanol, and dry under vacuum. 5.2.2 Glass Functionalization

Despite being a silicon oxide, glass presents very few Si–OH groups at its surface and needs to be activated, often by acidic treatment, before grafting with hybrid silylated peptides. Antibacterial bioorganic silica thin films have been obtained by dip coating in a solution containing a hybrid peptide displaying antibacterial properties [15]. The protected Si(OEt)3-(CH2)3-CO-AhxArg(Pbf)Arg (Pbf)-NH2 hybrid peptide has been immobilized on a glass slide, and protecting groups were removed by acidic treatment afterward. In that case, TEOS was used as a cement to create a hybrid peptidesilica layer of 100 nm. Strictly speaking, this is not a grafting, but a direct synthesis of a hybrid silica-peptide material on the top of the glass surface.

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Fig. 18 Glass functionalization with hybrid peptides Protocol 15 [15]: Example of Glass Functionalization with a Hybrid Peptide (Fig. 18)

1. Prepare a solution of TEOS/EtOH/H2O/HCl (1/73/8.3/0.01% mol). 2. Add the hybrid peptide (5 mol% related to TEOS) in the solution. 3. Stir for 5 h to allow hydrolysis of alkoxysilane units. 4. Clean the glass slide with ethanol by sonication, and activate by piranha treatment (H2SO4/H2O2, 70/30 v/v). 5. Drop off a thin film of the hybrid solution by dip coating at a constant withdrawal velocity of 14 cm/min. 6. Dry at 100  C for 24 h. 7. Treat with TFA/methylene chloride (1/1 v/v) to remove peptideprotecting groups. 8. Dry at 100  C for 24 h. 5.2.3 Titanium Functionalization

A different strategy is used to functionalize titanium. Indeed, the Si–O–Ti bond is too weak and unstable and can hydrolyze easily, and the direct functionalization of titanium oxide with hybrid peptides proved to be unsuccessful. Thus, a thin layer of silica coating is dropped off on the titanium substrate by dip coating before hybrid peptide immobilization.

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Silylated temporin-SHa amphipathic helical antimicrobial sequence has been used to functionalize a titanium substrate previ˚ ) [8]. Si–O–Si ously dip-coated with TEOS (thickness 72.3  1.9 A bond formed between silica and the hybrid was stable upon incubation. This method allowed an easy and site-specific covalent attachment of unprotected peptides on titanium surfaces. To study the influence of the orientation of the bioactive peptide on its antibacterial activity, siloxane functions were introduced on five different sites of temporin-SHa (N-terminus, C-terminus, and three side chains). It was shown that the temporin derivative immobilized through the middle of its sequence via a lysine displayed the better antibacterial activity. This feature was explained because the temporin derivative kept its overall net charge value (+2) and the alternate between polar and non-polar residues that is crucial for an α-helical folding. In addition, this mode of linkage should facilitate the lateral presentation of the hybrid peptide, with the hydrophobic face of the helix orientated toward the bacteria. Protocol 16 [8]: Example of Titanium Functionalization with a Hybrid Peptide

1. Clean the titanium substrate with ethanol by sonication, and activate with piranha treatment (H2SO4/H2O2, 70/30 v/v). 2. Drop off a silica layer by dip coating in an acidic (pH 1.5) colloidal solution of TEOS. 3. Dissolve the hybrid peptide in trifluoroethanol (TFE)/H2O/HCl 37% (64/2/0.1 v/v/v). 4. Incubate the plate in a 1.3 mM solution of the hybrid peptide for 24 h at 100  C. 5. Rinse the grafted plates with TFE and with distilled water. 5.3 Functionalization of Silica NPs

Silica nanoparticles (SiNPs) are attractive because of their easy synthesis by the sol-gel process (e.g., Sto¨ber process), their size control, their biocompatibility, and their ability to encapsulate different types of cargo molecules. Interestingly, any silylated molecule can be covalently grafted inside the SiNPs during the synthesis (e.g., a hybrid fluorophore). SiNPs are not different from bulk silica materials for grafting peptide: the presence of Si–OH moieties on the surface of SiNPS allows the reaction with silylated peptides. Ciccione et al. have developed a method to obtain in a single step well-defined tuneable multifunctional SiNPs, presenting at their surface covalently linked multiple hybrid ligands [9]. They demonstrated that it was possible to control and tune the ratio of grafted ligands simply by adjusting the relative concentration of hybrid species in the starting solution. An original fluorine nuclear magnetic resonance method was applied to the dissolved SiNPs to demonstrate this controlled grafting.

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Fig. 19 Functionalization of SiNPs by hybrid peptides Protocol 17 [9]: Example of SiNP Functionalization with Hybrid Peptides (Fig. 19)

1. Dissolve hybrid peptides 1 and 2 (2.5 mmol in total, the ratio being adjusted as desired) in 2 mL of a DMF/1% AcOH (v/v) mixture. 2. Add 100 mg of SiNPs (obtained by the Sto¨ber process) and stir for 12 h at 65  C. 3. Add 10 mL of DMF and centrifuge the solution at 33,500  g before removing the filtrate. 4. Repeat this procedure twice with DMF (10 mL) and twice with EtOH (10 mL). 5. Pour grafted SiNPs into 2 mL of demineralized water and freeze-dry. 6. Alternatively, NPs can be kept in solution (e.g., DPBS) to prevent aggregation.

6

Conclusion Hybrid silylated peptides are useful starting compounds for the design of functionalized materials. Their preparation can be performed using traditional peptide chemistry strategies, either in solution or on solid supports. Special care has to be paid to their isolation, as they are prone to premature condensation. Consequently, it is easier to engage hybrid peptides readily after their synthesis. The real strength of the hybrid peptides as building blocks lies in the fact that they can be handled like any other simple silane reagents, mixed in appropriate ratio and engaged in a sol-gel process. It enables a wide range of materials processing (e.g., thin films, nanoparticles, hydrogels, monoliths, xerogels, etc.) and a straightforward grafting on the surface of a metal oxide material (e.g., silica, titanium, silicone, glass) avoiding multistep conjugation reactions. Besides these applications, they unlock the

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possibility to craft novel bio-hybrid materials, whose synthesis cannot be envisioned by any other pathway, by using hybrid peptides as elementary functional bricks for supramolecular covalent assemblies in soft conditions. References 1. Montheil T, Echalier C, Martinez J, Subra G, Mehdi A (2018) Inorganic polymerization: an attractive route to biocompatible hybrid hydrogels. J Mater Chem B 6. https://doi. org/10.1039/C8TB00456K 2. Brinker CJ, Scherer GW (1990) Sol-gel science: the physics and chemistry of sol-gel processing. Gulf Professional Publishing, Houston, TX 3. Jebors S, Enjalbal C, Amblard M, Mehdi A, Subra G, Martinez J (2013) Bioorganic hybrid OMS by straightforward grafting of trialkoxysilyl peptides. J Mater Chem B 1(23):2921. https://doi.org/10.1039/c3tb20122h 4. Chauhan M, Chuit C, Corriu RJ, Mehdi A, Reye´ C (1996) Study of silyl cations bearing an aryldiamine pincer ligand. Organometallics 15(20):4326–4333 5. Marsmann H (1981) 29Si-NMR spectroscopic results. In: Kintzinger J-P, Marsmann H (eds) Oxygen-17 and Silicon-29, NMR basic principles and progress/NMR Grundlagen und Fortschritte. Springer, Berlin, Heidelberg, pp 65–235 6. Echalier C, Pinese C, Garric X, Van Den Berghe H, Jumas Bilak E, Martinez J, Mehdi A, Subra G (2016) Easy synthesis of tunable hybrid bioactive hydrogels. Chem Mater 28(5):1261–1265. https://doi.org/10. 1021/acs.chemmater.5b04881 7. Echalier C, Kalistratova A, Ciccione J, Lebrun A, Legrand B, Naydenova E, Gagne D, Fehrentz J-A, Marie J, Amblard M et al (2016) Selective homodimerization of unprotected peptides using hybrid hydroxydimethylsilane derivatives. RSC Adv 6 (39):32905–32914. https://doi.org/10. 1039/C6RA06075G 8. Masurier N, Tissot J-B, Boukhriss D, Jebors S, Pinese C, Verdie´ P, Amblard M, Mehdi A, Martinez J, Humblot V et al (2018) Sitespecific grafting on titanium surfaces with hybrid temporin antibacterial peptides. J Mater Chem B 6(12):1782–1790. https:// doi.org/10.1039/C8TB00051D 9. Ciccione J, Jia T, Coll J-L, Parra K, Amblard M, Jebors S, Martinez J, Mehdi A, Subra G (2016) Unambiguous and controlled

one-pot synthesis of multifunctional silica nanoparticles. Chem Mater 28(3):885–889. https://doi.org/10.1021/acs.chemmater. 5b04398 10. Jensen KJ, Alsina J, Songster MF, Va´gner J, Albericio F, Barany G (1998) Backbone Amide Linker (BAL) strategy for solid-phase synthesis of C-terminal-modified and cyclic peptides1,2,3. J Am Chem Soc 120 (22):5441–5452. https://doi.org/10.1021/ ja974116f 11. Jebors S, Ciccione J, Al-Halifa S, Nottelet B, Enjalbal C, M’Kadmi C, Amblard M, Mehdi A, Martinez J, Subra G (2015) A new way to silicone-based peptide polymers. Angew Chem Int Ed Engl 54(12):3778–3782. https://doi.org/10.1002/anie.201411065 12. Jedlicka SS, Little KM, Nivens DE, Zemlyanov D, Rickus JL (2007) Peptide ormosils as cellular substrates. J Mater Chem 17(48):5058. https://doi.org/10.1039/ b705393b 13. Jebors S, Pinese C, Nottelet B, Parra K, Amblard M, Mehdi A, Martinez J, Subra G (2015) Turning peptides in comb silicone polymers. J Pept Sci 21(3):243–247. https:// doi.org/10.1002/psc.2757 14. Martin J, Martinez J, Mehdi A, Subra G (2019) Silicone grafted bioactive peptides and their applications. Curr Opin Chem Biol 52:125–135 15. Jebors S, Cecillon S, Faye C, Enjalbal C, Amblard M, Mehdi A, Subra G, Martinez J (2013) From protected trialkoxysilyl-peptide building blocks to bioorganic–silica hybrid materials. J Mater Chem B 1(47):6510. https://doi.org/10.1039/c3tb21326a 16. Echalier C, Pinese C, van der Berghre H, Jumas-Bilak E, Martinez J, Mehdi A, Subra G (2016) Easy synthesis of tunable hybrid bioactive hydrogels. Chem Mater 28:1261–1265. https://doi.org/10.1021/acs.chemmater. 5b04881 17. Echalier C, Levato R, Mateos-Timoneda MA, ˜ o O, De´jean S, Garric X, Pinese C, Castan Noe¨l D, Engel E, Martinez J et al (2017) Modular bioink for 3D printing of biocompatible hydrogels: sol–gel polymerization of

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hybrid peptides and polymers. RSC Adv 7 (20):12231–12235. https://doi.org/10. 1039/C6RA28540F 18. Echalier C, Jebors S, Laconde G, Brunel L, Verdie´ P, Causse L, Bethry A, Legrand B, Van Den Berghe H, Garric X et al (2017) Sol-gel synthesis of collagen-inspired peptide hydrogel. Mater Today 20(2):59–66. https://doi. org/10.1016/j.mattod.2017.02.001 19. Maggini L, Travaglini L, Cabrera I, CastroHartmann P, De Cola L (2016) Biodegradable peptide-silica nanodonuts. Chem Eur J 22 (11):3697–3703. https://doi.org/10.1002/ chem.201504605 20. Papapostolou D, Smith AM, Atkins ED, Oliver SJ, Ryadnov MG, Serpell LC, Woolfson DN (2007) Engineering nanoscale order into a designed protein fiber. Proc Natl Acad Sci U S A 104(26):10853–10858 21. Branco MC, Schneider JP (2009) Selfassembling materials for therapeutic delivery. Acta Biomater 5(3):817–831 22. Go¨rbitz CH (2006) The structure of nanotubes formed by diphenylalanine, the core

recognition motif of Alzheimer’s B-amyloid polypeptide. Chem Commun (Camb) (22):2332–2334 23. Jebors S, Valot L, Echalier C, Legrand B, Mikhaleff R, Van Der Lee A, Amblard M, Martinez J, Mehdi A, Subra G (2019) Selfmineralization and assembly of a bis-silylated phe-phe pseudodipeptide to a structured bioorganic-inorganic material. Mater Horiz 6:2040–2046 24. Bhatt S, Pulpytel J, Arefi-Khonsari F (2015) Low and atmospheric plasma polymerisation of nanocoatings for bio-applications. Surf Innov 3(2):63–83. https://doi.org/10.1680/ sufi.14.00008 25. Pinese C, Jebors S, Stoebner PE, Humblot V, Verdie´ P, Causse L, Garric X, Taillades H, Martinez J, Mehdi A et al (2017) Bioactive peptides grafted silicone dressings: a simple and specific method. Mater Today Chem 4:73–83. https://doi.org/10.1016/j. mtchem.2017.02.007

Chapter 7 Synthesis of Peptide-Oligoethylene Glycol (OEG) Conjugates for Multivalent Modification of Nanomaterials Daniel Pulido and Miriam Royo Abstract Multivalent nanomaterials are designed to take advantage of the distinctive features that arise from the cooperativity of multiple “ligand-target” interactions, resulting in improved target affinities or enhanced cell/tissue targeting among others. Peptides are widely used biomolecules to create these nano-systems or to introduce function on them, either by formation of self-assembled structures or by their conjugation to other platforms/scaffolds. To circumvent the problems related to the stability of peptides in biological environments, one strategy is the peptide PEGylation. Traditionally, the use of high-molecular-weight PEG derivatives is the most common strategy, and the reported achievements certainly support this approach. However, depending on the desired applications, the polydispersity associated with these large-PEG derivatives becomes, occasionally, a serious drawback in terms of the reproducibility of biological behavior or the batch-to-batch consistency. Thus, in these scenarios, the use of shorter oligoethylene glycol (OEG) moieties, typically monodisperse compounds, is preferred to achieve nano-systems with more reproducible results. Herein we describe chemical methods to prepare (1) several OEG derivatives with different functional groups (amino, azido, thiol), (2) multivalent and multimodal OEG-based dendritic platforms conjugated to peptides, and (3) monovalent OEG-peptide conjugates with suitable chemical modifications used to create multivalent nanomaterials. Key words Oligoethylene glycol (OEG), Peptide conjugation, Multivalency, PEGylation, Nanomaterials, Dendritic platform, Solid-phase peptide synthesis

1

Introduction Peptides are highly relevant biomolecules in the field of biotechnological applications, playing an important role in the design and preparation of nanomaterials [1, 2]. These versatile compounds can be used per se to obtain self-assembled structured materials [3] or as functional components of other nano-systems of distinct natures [4]. As natural products, these biomolecules present diverse biological activities, as well as an excellent biocompatibility and, in the case of short peptide sequences, usually low degrees of immunogenicity when used in vivo. Among the different potential

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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uses of peptides, they have been reported as therapeutic agents [5, 6], as targeting moieties to specific cell types or tissues [7], or as three-dimensional structural scaffolds for tissue engineering or cell cultures. However, one major drawback of the use of peptides in bio-related applications is also associated with their nature, since they are natural substrates of enzymes which degrade them. In some cases, their half-life is in the range of few minutes, a major hurdle to exert any desired biological effect [8]. Traditionally, one of the approaches to circumvent this disadvantage consists in the “protection” of peptides by conjugation to other synthetic polymers to help overcome their intrinsic limitations related to proteolytic stability or in vivo circulation time. Polyethylene glycol (PEG) is usually one of the preferred candidates to address this issue. Conjugation of the hydrophilic polymer PEG to therapeutics (PEGylation) [9] has been traditionally a strategy to improve their safety and efficacy, by influencing positively on parameters such as solubility, stability, circulation time, toxicity, or immunogenicity. Currently, there are several successful examples of FDA-approved PEGylated drugs, mainly PEG-proteins [10]. Therefore, the abovementioned benefits of PEG conjugation have also been considered for the design of many peptide-based or peptide-containing nanomaterials. The variety of commercially available PEG-reagents used for conjugation is very wide, with different reactive moieties according to the desired chemical linkage and also variable molecular weights, going from few hundred Da to several kDa reagents. High-molecular-weight PEGs are typically polydisperse molecules, and from the point of view of the final conjugates, this broad distribution of molar mass may hinder the characterization of compounds, affect biological activity, or entail problems associated with the batch-to-batch reproducibility. On the other hand, polymers containing a reduced number of ethylene glycol repeats, namely, oligoethylene glycol (OEG) moieties (MW < ~2000 Da), are easier to be obtained as monodisperse compounds. In general, it is well accepted the use of both polydisperse and monodisperse PEGs to prepare PEG-peptide conjugates, but some advantages of monodisperse moieties are a major synthetic control of the resulting products and a more predictable/reproducible biological behavior, especially in vivo. Multivalent interactions are one of nature’s resources to regulate some biological processes, and they are the result of the cooperativity of multiple “ligand-target” interactions [11]. Accordingly, a large number of synthetic multivalent nanomaterials have been described with the aim to improve parameters such as target affinity, receptor clustering, or cell/tissue targeting. Traditionally, among other biomolecules, peptides have been incorporated as ligands in these nanomaterials to promote these multivalent interactions, and considering their aforementioned inherent limitations, they usually have been introduced in conjugation with PEG (or OEG) moieties.

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PEG is a biocompatible and flexible polymer that can contribute to minimize the steric hindrance and favor the accessibility of the multiple copies of peptides to target ligands. Furthermore, the presence of the polymer may also prevent an undesired early degradation of these peptides. The preparation of multivalent nanomaterials containing PEG-peptide conjugates can be addressed from two differentiated strategies. In the first scenario, a single chemical platform contains multiple PEG-peptide moieties; thus, the platform itself is multivalent, and it can be used either directly or incorporated to other nanomaterials for their modification. Examples of this are PEGylated peptide dendrimers used as targeted delivery vehicles for antitumor drugs [12] or star-shaped four-arm PEG scaffolds functionalized with peptides to modulate hydrogel properties [13] (thermal stabilities, rheological properties) or also as drug delivery vehicles [14]. In the second scenario, on the other hand, the PEG-peptide component is monovalent, and the multivalency is achieved after supramolecular self-assembling processes, as micellization [15] or liposome formulation [16], or after the conjugation of several copies of this conjugate to other biomaterial’s surface for its functionalization [17]. Regardless of the strategy used, from the point of view of the chemical bonds used to link both components, although there are many examples described, the most used ones are the amide bond, typically between a carboxy-OEG derivative and an amino group from the peptide, and the thioether bond, formed between a maleimide-OEG and a thiol-containing peptide. In this chapter we describe examples of OEG-peptide conjugates developed in our laboratory using the two previously described approaches to generate multivalent nano-systems, by means of inherently multivalent platforms or by monovalent conjugates that act as building blocks of other supramolecular structures. The general structure of the OEG derivatives and some examples of peptides used to prepare the different multivalent nano-systems are shown in Fig. 1, and the structure of the multivalent platforms that we have described is shown in Fig. 2. These dendritic compounds are composed of a synthetic derivative (1 or 2) of diethylenetriaminepentaacetic acid as the central core, which is functionalized with five monodisperse OEG moieties by amide bonds. Our methodology allows the derivatization of the surface functional groups in controlled ratios [18–20], and herein we describe the synthesis of the monomodal compound with five equivalent positions (13) and two examples of bimodal patterns, the 3-2 (14) and the 4-1 (15 and 16) ratios, respectively. Different peptides have been conjugated to these platforms and used, among others, to functionalize hydrogels with integrin binding peptides [21] or with affinity binding peptides for a controlled release of growth factors [22] and also to study their ability to chelate gadolinium ions to act as MRI contrast agents [23]. Conjugation of the

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Fig. 1 General structure of OEG derivatives (3–8) and peptides (9–12) used to prepare multivalent nanosystems

targeting peptides via amide bonds in compounds 17–19 (Fig. 2) is also described in detail. Alternatively, monovalent OEG-peptide conjugates (Fig. 3) have to be functionalized in the opposite end of the OEG chain with a precise moiety that allows the generation of multivalency either by self-assembling or surface conjugation. Herein we describe a cholesterol-OEG-peptide (20) as a component for liposome formulations [24, 25] and a thiol-OEG-peptide (21), used for monolayer functionalization (Fig. 3). In the former case the peptide is linked via a carbamate bond and in the latter by the formation of an amide. By following similar procedures for modification of OEG derivatives and using other peptide sequences, many different types of derivatives could also be afforded to generate/functionalize a large variety of versatile multivalent nanomaterials.

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Fig. 2 General structure of multivalent platforms (13–19) and DTPA derivatives (1–2) used to prepare them

2

Materials

2.1 General Laboratory Equipment, Solvents, and Solutions

1. Rotary evaporator. 2. High-performance liquid chromatography (HPLC) system equipped with analytical reversed-phase C18 or C4 columns (see Note 1).

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Fig. 3 Monovalent conjugates Cholesterol-OEG-cyclo(RGDfK) (20) and RGDS-OEG-thiol (21) used to generate multivalent systems by self-assembling or by surface conjugation

3. Lyophilizer. 4. Nuclear magnetic resonance (NMR) spectrometer. 5. Centrifuge. 6. Mass spectrometer (see Note 2). 7. CombiFlash® Companion® flash chromatography system on normal phase silica gel cartridges (ISCO). 8. Sonicator bath. 9. Desiccator.

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10. Acetonitrile (CH3CN). 11. Dichloromethane (CH2Cl2). 12. Methanol (MeOH). 13. Ethanol (EtOH). 14. N,N-Dimethylformamide (DMF). 15. Ethyl acetate (AcOEt). 16. Diethyl ether (Et2O). 17. n-Hexane. 18. Dioxane. 19. Chloroform (CHCl3). 20. Saturated aqueous sodium bicarbonate solution (NaHCO3). 21. Saturated aqueous sodium chloride solution (brine). 22. 1 M HCl aqueous solution. 23. Aqueous citric acid solution (0.5%, w/v). 24. Method 1 HPLC elution buffer A: 0.1% (v/v) HCOOH in water. 25. Method 1 HPLC elution buffer B: 0.07% (v/v) HCOOH in acetonitrile. 26. Method 2 HPLC elution buffer A: 0.045% (v/v) trifluoroacetic acid (TFA) in water. 27. Method 2 HPLC elution buffer B: 0.036% (v/v) TFA in acetonitrile. 2.2 Synthesis of Multivalent OEG Platforms

1. Ethanolamine. 2. tert-Butyl bromoacetate. 3. Potassium hydrogencarbonate (KHCO3). 4. Triphenylphosphine (PPh3). 5. N-Bromosuccinimide (NBS). 6. Glycine benzyl ester p-toluenesulfonate salt. 7. Phosphate buffer solution: 2 M PBS at pH 8. 8. Hydrogen chloride solution (HCl): 4 M hydrogen chloride in dioxane. 9. Sodium hydroxide solution (NaOH): 2 N aqueous sodium hydroxide. 10. Diethylenetriaminepentaacetic dianhydride).

dianhydride

(DTPA

11. 1-(tert-Butoxycarbonylamino)-4,7,10-trioxa-13-tridecanamine (3). 12. O-(2-Aminoethyl)-O0 -(2-azidoethyl)pentaethylene glycol (7).

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13. 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate (HBTU). 14. N,N-Diisopropylethylamine (DIEA). 15. Triethylamine (Et3N). 16. Imidazole-1-sulfonyl azide hydrochloride (see Note 3). 17. 3-(Tritylthio)propionic acid. 18. Potassium carbonate (K2CO3). 19. Di-tert-butyl dicarbonate (Boc2O). 20. Benzyl chloroformate. 21. Zinc powder. 22. Ammonium chloride (NH4Cl). 23. Palladium (10%, w/w) on activated carbon (Pd/C catalyst). 24. Hydrogen gas. 25. Celite®. 26. Waters PoraPak™ Rxn column. 27. Basic alumina for column chromatography. 28. Magnesium sulfate (MgSO4). 2.3 Solid-Phase Synthesis of Peptide Moieties

1. Standard N-α-Fmoc-amino acid building blocks for peptide synthesis. The side chains are protected by tert-butyl (tBu) for Asp, Glu, Thr, Tyr, and Ser; trityl (Trt) for Asn, Gln, His, and Cys; tert-butoxycarbonyl (Boc) for Lys and Trp; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg. 2. Polypropylene syringes fitted with a porous polyethylene porous disk filter for manual solid-phase peptide synthesis (SPPS). 3. Orbital shaker. 4. 2-Chlorotrityl chloride resin (100–200 mesh), 1% DVB, loading 1.0–1.6 mmol/g. 5. N,N0 -Diisopropylcarbodiimide (DIC). 6. Benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP). 7. Ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure). 8. 1-Hydroxybenzotriazole hydrate (HOBt·H2O). 9. Propylphosphonic anhydride solution, 50% weight in ethyl acetate (T3P). 10. N,N-Diisopropylethylamine (DIEA). 11. Triethylamine (Et3N).

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12. Kaiser test reagent A: Prepare a mixture of these two solutions: (a) 2 mL of 1 mM aqueous KCN and 98 mL of pyridine and (b) 40 g of phenol in 10 mL of ethanol. 13. Kaiser test reagent B: Dissolve 5 g of ninhydrin in 100 mL of ethanol. 14. Fmoc group removal solution: 20% (v/v) piperidine in DMF. 15. Cleavage solution A (fully deprotected peptides): Trifluoroacetic acid (TFA)/water/triisopropyl silane (TIS) (95:2.5:2.5, v/v/v). 16. Cleavage solution B (fully side-chain protected peptides): Trifluoroacetic acid (TFA)/dichloromethane (1:99, v/v). 17. Cleavage solution C (fully side-chain protected peptides): Acetic acid/trifluoroethanol/dichloromethane (1:3:3, v/v/v). 18. Di-tert-butyl dicarbonate (Boc2O). 2.4 Conjugation of Peptides to Multivalent Platforms. Synthesis of Compounds 17–19

1. Trifluoroacetic acid (TFA). 2. Benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP). 3. Cleavage solution A (fully deprotected peptides): Trifluoroacetic acid (TFA)/water/triisopropyl silane (TIS) (95:2.5:2.5, v/v/v). 4. N,N-Diisopropylethylamine (DIEA). 5. Regenerated cellulose dialysis membrane with MWCO 1 kDa.

2.5 Synthesis of Monovalent OEG-Peptide Platforms (20–21)

1. Cholesterol. 2. p-Toluenesulfonyl chloride. 3. Pyridine. 4. Tetraethylene glycol. 5. Phosgene solution ~20% in toluene. 6. N-Hydroxysuccinimide. 7. N,N-Diisopropylethylamine (DIEA). 8. Glass funnel filter with sintered glass disc with 10–16 μm pore size. 9. N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide chloride (EDC·HCl).

hydro-

10. 1-Hydroxybenzotriazole hydrate (HOBt·H2O). 11. Magnesium sulfate (MgSO4). 12. Cleavage solution A (fully deprotected peptides): Trifluoroacetic acid (TFA)/water/triisopropyl silane (TIS) (95:2.5:2.5, v/v/v).

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Methods

3.1 Multivalent OEG Platforms 3.1.1 Preparation of 4Benzyloxycarbonylmethyl1,1,7,7-;tetra (carboxymethyl)1,4,7-triazaheptane trihydrochloride (2) (Fig. 4) Preparation of 2-[Bis-(tertbutoxycarbonylmethyl) amino]ethyl bromide

1. Add KHCO3 (4.10 g, 41.0 mmol, 2.5 equiv) to a stirred solution of tert-butyl bromoacetate (5.3 mL, 35.9 mmol, 2.2 equiv) in DMF (50 mL), and cool the resulting suspension at 0  C. 2. Add ethanolamine (1.0 g, 16.4 mmol, 1.0 equiv) via syringe over a 5 min period and stir 30 min at 0  C. 3. Allow the reaction mixture to warm to room temperature (RT) and stir 16 h. 4. Remove DMF to dryness in a rotary evaporator under reduced pressure. 5. Add 50 mL of saturated aqueous NaHCO3 to the crude and extract with diethyl ether (3  50 mL). Wash the combined organic phases with brine (1  50 mL), dry the organic phase with MgSO4, and remove the solvent under reduced pressure. 6. Dissolve the crude in CH2Cl2 (50 mL), add solid triphenylphosphine (5.59 g, 21.3 mmol, 1.3 equiv), and cool the resulting solution at 0  C. 7. Add solid N-bromosuccinimide (3.79 g, 21.3 mmol, 1.3 equiv) and stir 2 h at 0  C. 8. Evaporate the solvent under reduced pressure and triturate the resulting crude with diethyl ether (50 mL). Filter the solid by vacuum and discard it. Evaporate the filtrate to dryness (see Note 4). 9. Purify the crude by flash column chromatography on silica using Et2O and hexane as solvents (5–30% Et2O in hexane).

Fig. 4 Synthesis of 4-benzyloxycarbonylmethyl-1,1,7,7-tetra(carboxymethyl)-1,4,7-triazaheptane trihydrochloride (2)

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10. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. 2-[Bis-(tert-butoxycarbonylmethyl)amino]ethyl bromide (4.22 g, 73%). 1H NMR (400 MHz, CDCl3, 298 K): δ 3.48 (s, 4H), 3.43 (t, J ¼ 7.6 Hz, 2H), 3.13 (t, J ¼ 7.6 Hz, 2H), 1.46 (s, 18H). 13C NMR (101 MHz, CDCl3, 298 K): δ 170.7, 81.4, 56.8, 56.6, 30.4, 28.3. ESI-MS (positive mode) m/z calcd. For C14H27BrNO4 [M+H]+ 352.1; found 352.2. Alkylation of Glycine Benzyl Ester

1. Prepare a mixture of glycine benzyl ester p-toluenesulfonate salt (1.06 g, 3.14 mmol, 1.0 equiv) and bromoderivative described in Subheading “Preparation of 2-[Bis-(tert-butoxycarbonylmethyl)amino]ethyl bromide” (2.31 g, 6.56 mmol, 2.1 equiv) in acetonitrile (50 mL). 2. Add a 2 M phosphate buffer solution at pH 8 (50 mL). 3. Stir vigorously 24 h at RT (see Note 5). 4. Remove the acetonitrile by evaporation and extract the aqueous phase with CH2Cl2 (3  50 mL). 5. Wash the combined organic phases with brine (1  100 mL), dry the organic phase with MgSO4, and remove the solvent under reduced pressure. 6. Purify the crude by column chromatography on basic alumina using hexane and ethyl acetate as solvents (5–20% ethyl acetate in hexane). 7. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. 4-Benzyloxycarbonylmethyl-1,1,7,7-tetra(tert-butoxycarbonylmethyl)-1,4,7-triazaheptane (1.42 g, 64%). 1H NMR (400 MHz, CDCl3, 298 K): δ 7.37–7.29 (m, 5H), 5.12 (s, 2H), 3.62 (s, 2H), 3.42 (s, 8H), 2.83 (s, 8H), 1.44 (s, 36H). 13 C NMR (101 MHz, CDCl3, 298 K): δ 171.5, 170.7, 136.0, 128.6, 128.4, 128.3, 81.0, 66.1, 56.2, 55.0, 52.9, 52.4, 28.3. ESI-MS (positive mode) m/z calcd. For C37H62N3O10 [M +H]+ 708.4; found 708.4.

Removal of Tert-Butyl-Protecting Groups

1. Dissolve the product from Subheading “Alkylation of Glycine Benzyl Ester” (1.03 g, 1.45 mmol) in a 4 M HCl/dioxane solution (10 mL), and stir the mixture 3 h at 80  C. 2. Evaporate the mixture to dryness in a rotary evaporator and coevaporate two times with 10 mL of dioxane. 3. Dissolve the crude in a minimal volume of H2O and load into a 10 g Waters PoraPak™ Rxn column (see Note 6). 4. Elute the column with H2O (100 mL).

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5. Elute with H2O:CH3CN (100 mL, 97:3 v/v, 0.1% HCOOH) and then with H2O:CH3CN (90:10 v/v, 0.1% HCOOH) until no product is detected by HPLC. 6. Monitor the collected fractions by HPLC and combine those containing the pure product. 7. Evaporate the CH3CN in a rotary evaporator and lyophilize the aqueous phase. 8. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. 4-Benzyloxycarbonylmethyl-1,1,7,7-tetra(carboxymethyl)-1,4,7-triazaheptane trihydrochloride salt (765 mg, 89%). 1H NMR (400 MHz, D2O, 298 K): δ 7.49–7.42 (m, 5H), 5.24 (s, 2H), 4.12 (s, 8H), 3.70 (s, 2H), 3.49 (t, J ¼ 6.0 Hz, 4H), 3.13 (t, J ¼ 6.0 Hz, 4H). 13C NMR (101 MHz, D2O, 298 K): δ 172.3, 169.3, 135.4, 129.1, 128.9, 66.7, 55.7, 54.2, 53.4, 49.6. HRMS ESI (positive mode) m/z calcd. For C21H30N3O10 (amine) [M+H]+ 484.1926; found 484.1920. 3.1.2 Preparation of OEG Derivatives (4–6, 8) 1-(Benzyloxycarbonylamino)-4,7,10trioxa-13-tridecanamine (4)

1. Add benzyl chloroformate (0.67 mL, 4.69 mmol, 1.5 equiv) and Et3N (1.30 mL, 9.33 mmol, 3.0 equiv) to a stirred solution of 1-(tert-butoxycarbonylamino)-4,7,10-trioxa-13-tridecanamine (3) (1.00 g, 3.12 mmol, 1.0 equiv) in CH2Cl2 (5 mL), and stir 1 h at RT. 2. Pour the reaction mixture into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (2  50 mL), aqueous citric acid solution (0.5%, w/v) (2  50 mL), and brine (1  50 mL). 3. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 4. Purify the crude by flash column chromatography on silica using CH2Cl2 and MeOH as solvents (0–5% MeOH in CH2Cl2). 5. Dissolve the obtained pure product in a 4 M HCl/dioxane solution (3 mL) and stir the mixture 1 h at RT. 6. Evaporate the mixture to dryness in a rotary evaporator. 7. Dissolve the crude in 2 N NaOH (20 mL), pour the solution into a separation funnel, and extract with CH2Cl2 (6  20 mL). 8. Dry the combined organic phases with MgSO4 and remove the solvent under reduced pressure. 9. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. 1-(Benzyloxycarbonylamino)-4,7,10-trioxa-13-tridecanamine (818 mg, 74%). 1H NMR (400 MHz, CDCl3, 298 K): δ

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7.37–7.27 (m, 5H), 5.68 (bs, NH), 5.08 (s, 2H), 3.65–3.47 (m, 12H), 3.34–3.26 (m, 2H), 2.81 (t, J ¼ 6.8 Hz, 2H), 1.83–1.67 (m, 4H). 13C NMR (101 MHz, CDCl3, 298 K): δ 156.7, 136.9, 128.5, 128.1, 128.0, 70.6, 70.6, 70.2, 70.2, 69.5, 66.5, 39.5, 39.1, 32.4, 29.5. ESI-MS (positive mode) m/z calcd. For C18H31N2O5 [M+H]+ 355.2; found 355.3. 1-Azido-4,7,10trioxa-13-tridecanamine hydrochloride (5)

1. Add K2CO3 (2.03 g, 14.7 mmol, 1.05 equiv) to a stirred solution of 1-(tert-butoxycarbonylamino)-4,7,10-trioxa-13tridecanamine (3) (4.50 g, 14.0 mmol, 1.0 equiv) in a mixture of H2O and MeOH (90 mL, 1:1 (v/v)), and stir at RT to the complete dissolution of the K2CO3. 2. Add imidazole-1-sulfonyl azide hydrochloride (2.93 g, 14.0 mmol, 1.0 equiv) and stir 20 min at RT. 3. Add extra K2CO3 (2.03 g, 14.7 mmol, 1.05 equiv) and stir 25 min at RT (see Note 7). 4. Remove the MeOH by evaporation, pour the resulting aqueous phase into a separation funnel, and extract with CH2Cl2 (3  75 mL). 5. Wash the combined organic phases with aqueous citric acid solution (0.5%, w/v) (2  75 mL) and brine (1  75 mL), dry with MgSO4, and remove the solvent under reduced pressure. 6. Purify the crude by flash column chromatography on silica using CH2Cl2 and EtOH as solvents (0–5% EtOH in CH2Cl2). 7. Dissolve the obtained pure product in a 4 M HCl/dioxane solution (10 mL) and stir the mixture 1 h at RT. 8. Evaporate the mixture to dryness in a rotary evaporator, dissolve the product in H2O, and lyophilize. 9. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. 1-Azido-4,7,10-trioxa-13-tridecanamine hydrochloride (3.49 g, 88%). 1H NMR (400 MHz, CDCl3, 298 K): δ 8.15 (bs, 3H), 3.70–3.60 (m, 10H), 3.58 (t, J ¼ 6.1 Hz, 2H), 3.41 (t, J ¼ 6.7 Hz, 2H), 3.19 (bs, 2H), 2.10–2.02 (m, 2H), 1.92–1.84 (m, 2H). 13C NMR (101 MHz, CDCl3, 298 K): δ 70.5, 70.3, 70.2, 70.2, 70.1, 69.6, 48.6, 39.2, 29.2, 26.9. ESI-MS (positive mode) m/z calcd. For C10H23N4O3 [M +H]+ 247.2; found 247.0.

1-(3-(Tritylthio)propanoylamino)-4,7,10trioxa-13-tridecanamine (6)

1. Dissolve 3-(tritylthio)propionic acid (250 mg, 0.72 mmol, 1.0 equiv), EDC·HCl (206 mg, 1.07 mmol, 1.5 equiv), and HOBt·H2O (165 mg, 1.08 mmol, 1.5 equiv) in DMF (3 mL).

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2. Add a solution of 1-azido-4,7,10-trioxa-13-tridecanamine hydrochloride (5) (214 mg, 0.76 mmol, 1.05 equiv) and DIEA (245 μL, 1.41 mmol, 2.0 equiv) in DMF (2 mL). 3. Stir the mixture 6 h at RT (see Note 8). 4. Evaporate the mixture to dryness in a rotary evaporator. 5. Dissolve the crude in CH2Cl2 (20 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (3  20 mL), aqueous citric acid solution (0.5%, w/v) (3  20 mL), and brine (1  20 mL). 6. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure to obtain an intermediate product (397 mg, 96%) with a trityl-protected thiol in one of the OEG terminal positions and an azido group in the other. 7. Dissolve the intermediate product (194 mg, 0.34 mmol, 1.0 equiv) in MeOH (15 mL), and add Pd/C catalyst (20 mg, 10% w/w of the total product). 8. Purge the suspension with N2 and set it under a H2 atmosphere (1 bar). Stir 3 h at RT. 9. After exposure to H2, filter the reaction mixture through Celite® and concentrate to dryness in a rotary evaporator. 10. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. 1-(3-(Tritylthio)propanoyl-amino)-4,7,10-trioxa-13-tridecanamine (169 mg, 91%, global yield 87%). 1H NMR (400 MHz, CDCl3, 298 K): δ 7.43–7.37 (m, 6H), 7.30–7.23 (m, 6H), 7.22–7.16 (m, 3H), 6.46 (br t, J ¼ 5.2 Hz, NH), 3.63–3.47 (m, 12H), 3.32–3.24 (m, 2H), 2.82 (t, J ¼ 6.5 Hz, 2H), 2.46 (t, J ¼ 7.4 Hz, 2H), 2.10 (t, J ¼ 7.4 Hz, 2H), 1.78–1.68 (m, 4H). 13C NMR (101 MHz, CDCl3, 298 K): δ 171.1, 144.9, 129.7, 129.7, 128.0, 126.8, 70.6, 70.5, 70.2, 70.1, 69.8, 69.7, 66.8, 39.8, 37.6, 35.5, 32.3, 29.0, 27.9. ESI-MS (positive mode) m/z calcd. For C32H43N2O4S [M+H]+ 551.3; found 551.5. O-(2-Aminoethyl)O0 -[(2-(Boc-amino)ethyl] pentaethylene glycol (8)

1. Add DIEA (0.60 mL, 3.44 mmol, 1.2 equiv) and di-tert-butyldicarbonate (674 mg, 3.09 mmol, 1.1 equiv) to a solution of O-(2-aminoethyl)-O0 -(2-azidoethyl)pentaethylene glycol (7) (1.00 g, 2.86 mmol, 1.0 equiv) in CH2Cl2 (50 mL), and stir 1 h at RT. 2. Pour the reaction mixture into a separation funnel and wash the organic layer with saturated aqueous NaHCO3 (3  50 mL) and brine (1  50 mL). 3. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure.

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4. Dissolve the product in a mixture of H2O and EtOH (10 mL, 3:7 (v/v)), add NH4Cl (350 mg, 6.54 mmol, 2.3 equiv) and zinc powder (240 mg, 3.67 mmol, 1.3 equiv), and stir at RT until the reaction reaches completion (monitor by HPLC) (see Note 9). 5. Evaporate the solvent under reduced pressure and add CH3CN (40 mL) to the resulting crude. Filter the solid by vacuum and discard it. Evaporate the filtrate to dryness. 6. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. O-(2-Aminoethyl)-O0 -[(2-(Boc-amino)ethyl]pentaethylene glycol (900 mg, 74%). 1H NMR (400 MHz, CDCl3, 298 K): δ 3.67–3.58 (m, 22H), 3.54 (t, J ¼ 5.1 Hz, 2H), 3.40 (t, J ¼ 4.4 Hz, 2H), 3.30 (bs, 2H), 1.44 (s, 9H). 13C NMR (101 MHz, CDCl3, 298 K): δ 156.5, 79.2, 70.1, 70.07, 70.05, 70.01, 69.9, 69.8, 66.8, 40.2, 28.4. ESI-MS (positive mode) m/z calcd. For C19H41N2O8 [M+H]+ 425.3; found 425.7. 3.1.3 Synthesis of Monomodal Platform 13 (Fig. 5)

1. To a suspension of DTPA dianhydride (1) (25 mg, 70.0 μmol, 1.0 equiv) in DMF (5 mL), add DIEA (97 μL, 0.557 mmol, 8.0 equiv) and HBTU (106 mg, 0.279 mmol, 4.0 equiv). Stir the mixture 2 min at RT. 2. Add a solution of 1-(tert-butoxycarbonylamino)-4,7,10trioxa-13-tridecanamine (3) (135 mg, 0.42 mmol, 6.0 equiv) in DMF (3 mL), and stir 1 h at RT. 3. Evaporate the mixture to dryness in a rotary evaporator. 4. Dissolve the crude in AcOEt (15 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (3  15 mL), 1 M HCl aqueous solution (3  15 mL), and brine (1  15 mL). 5. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 6. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. Monomodal platform 13 (126 mg, 94%). 1H NMR (400 MHz, CDCl3, 298 K): δ 7.68 (bs, NH), 7.44 (bs, NH), 5.09 (bs, NH), 3.67–3.55 (m, 42H), 3.53 (t, J ¼ 6.0 Hz, 20H), 3.38–3.30 (m, 10H), 3.26–3.14 (m, 18H), 3.08 (s, 2H), 2.71–2.55 (m, 8H), 1.85–1.70 (m, 20H), 1.43 (s, 45H); 13C NMR (101 MHz, CDCl3, 298 K): δ 170.8, 156.2, 79.0, 70.6, 70.6, 70.3, 70.3, 69.5, 69.5, 59.3, 58.7, 53.5, 53.3, 38.5, 37.2, 29.8, 29.5, 28.6. HRMS ESI (positive mode) m/z calcd. for C89H174N13O30 [M+H]+ 1905.2484; found 1905.2471.

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Fig. 5 Synthesis of multivalent platforms 13–16 3.1.4 Synthesis of Bimodal Platform 14 (Fig. 5)

1. Add a solution of 1-(benzyloxycarbonylamino)-4,7,10-trioxa13-tridecanamine (4) (723 mg, 2.04 mmol, 2.0 equiv) in dry DMF (10 mL) to a 50 mL round-bottom flask containing solid DTPA dianhydride (1) (364 mg, 1.02 mmol, 1.0 equiv) and stir the mixture 1 h at RT. 2. Evaporate the mixture to dryness in a rotary evaporator. 3. Dissolve the crude in CH2Cl2 (75 mL), pour it into a separation funnel, and wash the organic layer with a 1:1 (v/v) mixture of aqueous citric acid solution (0.5%, w/v) and brine (3  75 mL).

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4. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure to obtain an intermediate product (1.00 g, 92%) with two of the five carboxylic acids of the core acylated with the OEG derivative. 5. Dissolve the intermediate product (672 mg, 630 μmol, 1.0 equiv) in CH2Cl2 (30 mL), and add DIEA (0.72 mL, 4.13 mmol, 6.6 equiv) and PyBOP (1.08 g, 2.08 mmol, 3.3 equiv) (see Note 10). Stir the mixture 2 min at RT. 6. Add a solution of 1-(tert-butoxycarbonylamino)-4,7,10trioxa-13-tridecanamine (3) (0.67 g, 2.09 mmol, 3.3 equiv) in CH2Cl2 (5 mL), and stir 1 h at RT. 7. Pour the reaction mixture into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (2  50 mL), aqueous citric acid solution (0.5%, w/v) (2  50 mL), and brine (1  50 mL) (see Note 11). 8. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 9. Dissolve the crude in CH2Cl2 (5 mL) and transfer to a centrifuge tube (50 mL). 10. Add hexane (40 mL), shake vigorously the mixture, and then centrifuge (5 min at 2200  g). Discard the supernatant. 11. Dissolve the oily precipitate in CH2Cl2 (5 mL) and repeat step 10 (see Note 12). 12. Dissolve the product in the minimal amount of a mixture of H2O and CH3CN (1:2 (v/v)) and lyophilize. 13. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. Bimodal platform 14 (994 mg, 80% from the intermediate, global yield 74%). 1H NMR (400 MHz, CDCl3, 298 K): δ 7.71 (bs, NH), 7.46 (bs, NH), 7.37–7.28 (m, 10H), 5.56 (bs, NH), 5.13 (bs, NH), 5.08 (s, 4H), 3.66–3.45 (m, 60H), 3.37–3.25 (m, 14H), 3.24–3.12 (m, 14H), 3.07 (s, 2H), 2.68–2.52 (m, 8H), 1.83–1.70 (m, 20H), 1.43 (s, 27H). 13C NMR (101 MHz, CDCl3, 298 K): δ 171.2, 170.9, 156.7, 156.2, 136.9, 128.6, 128.2, 128.1, 79.1, 70.6, 70.6, 70.6, 70.3, 70.3, 70.2, 69.6, 69.5, 69.4, 69.4, 66.6, 59.3, 58.7, 53.5, 53.3, 39.2, 38.5, 37.2, 37.0, 29.8, 29.7, 29.6, 29.5, 28.6. HRMS ESI (positive mode) m/z calcd. For C95H171N13O30 [M+2H]2+ 987.1122; found 987.1125. 3.1.5 Synthesis of Bimodal Platform 15 (Fig. 5)

1. To a solution of 4-benzyloxycarbonylmethyl-1,1,7,7-tetra(carboxymethyl)-1,4,7-triazaheptane trihydrochloride (2) (275 mg, 464 μmol, 1.0 equiv) in DMF (35 mL), add DIEA (1.21 mL, 6.96 mmol, 15 equiv) and HBTU (844 mg, 2.22 mmol, 4.8 equiv). Stir the mixture 2 min at RT.

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2. Add a solution of 1-(tert-butoxycarbonylamino)-4,7,10trioxa-13-tridecanamine (3) (743 mg, 2.32 mmol, 5.0 equiv) in DMF (3 mL), and stir 1 h at RT. 3. Evaporate the mixture to dryness in a rotary evaporator. 4. Dissolve the crude in AcOEt (25 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (3  25 mL), 1 M HCl aqueous solution (3  25 mL), and brine (1  25 mL). 5. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 6. Dissolve the product in MeOH (10 mL) and add Pd/C catalyst (10% w/w of the total crude product). 7. Purge the suspension with N2 and set it under a H2 atmosphere (1 bar). Stir 1 h at RT. 8. After exposure to H2, filter the reaction mixture through Celite®, and concentrate to dryness in a rotary evaporator to obtain an intermediate product (482 mg, 65%) with one free carboxylic acid and the other four acylated with the OEG derivative (see Note 13). 9. Dissolve the intermediate product (444 mg, 277 μmol, 1.0 equiv) in DMF (5 mL), and add DIEA (0.19 mL, 1.11 mmol, 4.0 equiv) and HBTU (131 mg, 346 μmol, 1.25 equiv). Stir the mixture 2 min at RT. 10. Add a solution of 1-azido-4,7,10-trioxa-13-tridecanamine hydrochloride (5) (97.9 mg, 346 μmol, 1.25 equiv) in DMF (2 mL), and stir 1 h at RT. 11. Evaporate the mixture to dryness in a rotary evaporator. 12. Dissolve the crude in AcOEt (30 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (3  30 mL), 1 M HCl aqueous solution (3  30 mL), and brine (1  30 mL). 13. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 14. Check the purity of the synthesized compound by 1H, 13C NMR, and MS. Bimodal platform 15 (478 mg, 94% from the intermediate, global yield 61%). 1H NMR (400 MHz, CDCl3): δ 7.60 (bs, NH), 7.37 (bs, NH), 5.06 (bs, NH), 3.64–3.44 (m, 60H), 3.39–3.24 (m, 12H), 3.23–3.08 (m, 16H), 3.04 (s, 2H), 2.66–2.50 (m, 8H), 1.85–1.66 (m, 20H), 1.39 (s, 36H). 13C NMR (101 MHz, CDCl3): δ 171.1, 170.7, 156.1, 78.9, 70.6, 70.5, 70.3, 70.2, 70.2, 69.5, 67.9, 59.2, 53.4, 53.3, 48.5, 38.5, 37.2, 29.8, 29.5, 29.1, 28.5. ESI-MS (positive mode)

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m/z calcd. For C84H164N15O28 [M+H]+ 1831.2; found 1832.2, m/z calcd. For C84H165N15O28 [M+2H]2+ 916.1; found 916.5. 3.2 Solid-Phase Peptide Synthesis

The peptides described in this chapter are designed with a Cterminal carboxylic acid moiety which is used either to couple the peptide to the oligoethylene glycol moiety or to perform the headto-tail cyclization to obtain the cyclic peptide. To this aim, the syntheses are carried out on the 2-chlorotrityl chloride resin using the Fmoc/tBu strategy.

3.2.1 General Procedures

The general protocols for the synthesis of peptides are described in the following Subheadings “Coupling of the First Amino Acid” to “Standard Coupling Procedure”. After the initial incorporation of the first amino acid to the resin (described in Subheading “Coupling of the First Amino Acid”), the iterative repetition of the steps of N-α-Fmoc removal (described in Subheading “Standard Fmoc Removal”) and amino acid coupling (described in Subheading “Standard Coupling Procedure”) affords the desired protected peptide sequence attached to the resin.

Coupling of the First Amino Acid

1. Place the desired amount of the resin in the syringe equipped with the filter. 2. Wash the resin (approximately 10 mL/g resin) thoroughly with DCM (3  1 min), DMF (3  1 min), and again with DCM (3  1 min). 3. Dissolve the Fmoc-protected amino acid (1.0–1.2 equiv, relative to the desired resin loading) and DIEA (5.0–8.0 equiv, relative to the initial resin loading) in the minimal amount of dry CH2Cl2 (see Note 14). 4. Add the mixture to the resin and stir for 90 min at RT in an orbital shaker. 5. To perform the capping of the remaining reactive groups, add methanol (0.8 mL/g resin) to the resin, and stir 20 min at RT. 6. Wash the resin (approximately 10 mL/g resin) with DCM (3  1 min), DMF (3  1 min), and again with DCM (3  1 min).

Determination of Resin Loading

1. Weight a small sample of dry resin (5–10 mg) in a syringe equipped with a porous disk filter (see Note 15). 2. Add 20% (v/v) piperidine in DMF (5–10 mL) to the resin and leave for 5 min at RT. 3. Filter the solution by vacuum and collect it in a 100 mL volumetric flask. 4. Repeat steps 2 and 3 (using the same volumetric flask).

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5. Add DMF to the volumetric flask to reach a 100 mL volume. 6. Measure the absorbance of the solution obtained at λ ¼ 301 nm (Abs301). 7. Calculate the loading according to the following equation: Loading ðmmol=gÞ ¼

Abs301  V  1000 , ε301  l  mresin

where V ¼ the volume of the volumetric flask in mL, ε301 ¼ 7800 M1 cm1, l ¼ cuvette size in cm, and mresin ¼ amount of resin in mg. Standard Fmoc Removal

1. Add 20% (v/v) piperidine in DMF (approximately 10 mL/g resin) to the resin. 2. Leave to react for 5–7 min at RT with occasional manual stirring using a Teflon bar. 3. Remove the mixture by vacuum filtration. 4. Repeat steps 1 and 2. 5. Remove the mixture by vacuum filtration and wash the resin with DMF (3  1 min) and DCM (3  1 min).

Standard Coupling Procedure

1. Dissolve the Fmoc-protected amino acid (3 equiv relative to the resin loading) and OxymaPure (or HOBt·H2O, see Note 16) (3 equiv relative to the resin loading) in the minimal amount of DMF. 2. Add DIC (3 equiv relative to the resin loading) and stir the mixture 1–2 min at RT (see Note 17). 3. Add the resulting solution to the resin and stir for 1 h at RT in an orbital shaker. 4. Wash the resin (approximately 10 mL/g resin) with DCM (3  1 min), DMF (3  1 min), and finally with DCM (3  1 min). 5. Check the completeness of the amino acid coupling by performing a qualitative Kaiser test (see Note 18). 6. In the case of incomplete amino acid incorporation, the coupling procedure is repeated.

3.2.2 Linear Protected Peptides (9–11)

The protected linear peptides with the terminal N-α-Fmoc group are prepared following the described general procedures (Subheading 3.2.1).

Introduction of Boc Group at N-terminal α-Amino Group

1. Remove the N-terminal Fmoc-protecting group as described in Subheading “Standard Fmoc Removal”. 2. Add to the resin a dissolution of Boc2O (3 equiv relative to the resin loading) and DIEA (6 equiv relative to the resin loading) in the minimal amount of CH2Cl2, and stir for 1 h at RT in an orbital shaker.

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3. Wash the resin (approximately 10 mL/g resin) with DCM (3  1 min), DMF (3  1 min), and finally with DCM (3  1 min). Peptide Cleavage

1. Add cleavage solution B to the resin (approximately 10 mL/g resin) and leave to react for 2 min at RT. 2. Filter the solution by vacuum and collect it in a flask containing H2O (1:1 (v/v) with respect to the final volume of cleavage solution) (see Note 19). 3. Repeat steps 1 and 2 four times (using the same flask to collect the filtrates). 4. Evaporate the combined filtrates in a rotary evaporator to remove the organic solvent. 5. Lyophilize the remaining aqueous suspension containing the fully protected peptide.

3.2.3 Cyclo(RGDfK) Peptide (12)

1. Add cleavage solution C to the resin (approximately 15 mL/g resin) and leave to react for 1 h at RT. 2. Filter the solution by vacuum and collect it in a flask.

Peptide Cleavage

3. Repeat steps 1 and 2 two times, reducing the time of reaction to 20–30 min (using the same flask to collect the filtrates). 4. Wash the resin two times with CH2Cl2 (15 mL/g resin). Collect the washings in the same flask with previous filtrates. 5. Evaporate the mixture to dryness in a rotary evaporator. 6. Coevaporate two times with toluene (25–30 mL).

Peptide Cyclization

1. Dissolve the linear peptide acetate salt in CH2Cl2 (approximately 85–100 mM). 2. Add the peptide solution to a flask containing a mixture of T3P (5.0 equiv) and Et3N (25 equiv) in CH2Cl2 (final concentration of peptide approximately up to 1.0–1.2 mM). 3. Stir the mixture at RT. 4. Monitor the complete disappearance of the linear peptide by HPLC. 5. Concentrate the mixture in a rotary evaporator to reduce the volume. 6. Pour the reaction mixture into a separation funnel and wash the organic layer two times with brine. 7. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure.

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Removal of Protecting Groups and Isolation of the Peptide

1. Add cleavage solution A to the crude (approximately 10–15 mL/g of peptide crude) and leave to react for 2–3 h at RT. 2. Concentrate the mixture in a rotary evaporator to reduce the volume to approximately one half-one third of the total. 3. Add cold Et2O (seven- to eightfold with respect to the remaining volume of cleavage solution). 4. Centrifuge the precipitate for 5 min at 2200  g at 4  C. 5. Decant the ether and wash the precipitate two times with additional cold Et2O. 6. Dissolve the solid in H2O/CH3CN (1:1 (v/v)) and lyophilize (see Note 20).

3.3 Synthesis of Multivalent Platforms 17–19

3.3.1 Removal of Boc-Protecting Group

The synthesis of these compounds is carried out using the same synthetic precursor, the multivalent platform 16. To prepare this product, follow the procedures described in Subheading 3.1.5 but using the OEG derivatives 8 and 7 as a replacement for OEG derivatives 3 and 5, respectively (see Figs. 1 and 5). 1. Dissolve platform 16 (scale 50–100 mg) in 5 mL TFA/H2O (95:5 (v/v)) and stir 1 h at RT. 2. Concentrate the mixture in a rotary evaporator to reduce the volume to approximately one half of the total. 3. Add cold Et2O (seven- to eightfold with respect to the remaining volume of cleavage solution). 4. Centrifuge the precipitate for 5 min at 2200  g at 4  C. 5. Decant the ether and wash the precipitate two times with additional cold Et2O.

3.3.2 Coupling of the Peptide

1. Dissolve the product obtained in Subheading 3.3.1 in the minimal amount of DMF, add PyBOP (4.4 equiv) and the linear protected peptide (4.4 equiv) (obtained from Subheading 3.2.2). 2. Adjust the pH of the mixture to 8 by addition of DIEA. 3. Stir the mixture at RT until the reaction reaches completion (monitor by HPLC). 4. Evaporate the mixture to dryness in a rotary evaporator. 5. Dissolve the crude in CH2Cl2 (20 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (3  20 mL) and brine (1  20 mL). 6. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure.

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7. Dissolve the crude in CH2Cl2 (5 mL) and transfer to a centrifuge tube (50 mL). 8. Add hexane (40 mL), shake vigorously the mixture, and then centrifuge (5 min at 2200  g). Discard the supernatant. 3.3.3 Removal of Protecting Groups and Isolation of the Peptide

1. Add cleavage solution A to the crude (approximately 10 mL/ 100–150 mg of crude) and leave to react for 2–3 h at RT (monitor the complete removal of protecting groups by HPLC). 2. Concentrate the mixture in a rotary evaporator to reduce the volume to approximately one half-one third of the total. 3. Add cold Et2O (seven- to eightfold with respect to the remaining volume of cleavage solution). 4. Centrifuge the precipitate for 5 min at 2200  g at 4  C. 5. Decant the ether and wash the precipitate two times with additional cold Et2O. 6. Dissolve the precipitate in water and dialyze against distilled water for 24 h at RT (1 kDa MWCO), and then lyophilize. 7. Check the purity of the synthesized compound by HPLC and MS. Multivalent platform 17 (52 mg, 58%). HPLC purity 95% (210 nm) (see Note 21). HRMS ESI (positive mode) m/z calcd. For C144H271N43O63 3610.9324; found 3610.9268. Multivalent platform 18 (57 mg, 45%). HPLC purity 90% (210 nm) (see Note 21). HRMS ESI (positive mode) m/z calcd. For C232H375N51O75 5075.7098; found 5075.7092. Multivalent platform 19 (130 mg, 75%). HPLC purity 91% (210 nm) (see Note 21). HRMS ESI (positive mode) m/z calcd. For C212H371N55O71 4823.7111; found 4823.7106.

3.4 Synthesis of Cholesteryl Tetraethylene Glycol-Cyclo(RGDfK) Conjugate (20) 3.4.1 Synthesis of Cholesteryl Tetraethylene Glycol

1. Dissolve cholesterol (1.50 g, 3.88 mmol, 1.0 equiv) in pyridine (10 mL) and cool the solution to 0  C in an ice bath. 2. Add a solution of p-toluenesulfonyl chloride (1.48 g, 7.76 mmol, 2.0 equiv) in pyridine (5 mL) at 0  C. 3. Remove the ice bath to allow the mixture to slowly warm to room temperature and stir overnight (16 h). 4. Evaporate the mixture to dryness in a rotary evaporator. 5. Dissolve the crude in CHCl3 (10 mL) and add it dropwise to a flask containing cold MeOH (80 mL). 6. Collect the precipitate by vacuum filtration using a glass funnel filter with sintered glass disc of 10–16 μm pore size. 7. Wash the precipitate two times with cold MeOH (25 mL) and one time with cold CH3CN (25 mL).

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8. Dry the solid in a vacuum desiccator overnight (1.53 g cholesteryl tosylate, 73%). 9. Add tetraethylene glycol (8.24 g, 42.4 mmol, 15 equiv) to a solution of cholesteryl tosylate (1.53 g, 2.83 mmol, 1.0 equiv) in dry dioxane (15 mL). 10. The mixture is heated to reflux (101  C) under Ar atmosphere and stirred for 6 h. 11. Evaporate the mixture to dryness in a rotary evaporator. 12. Dissolve the crude in CH2Cl2 (50 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (2  50 mL), H2O (2  50 mL), and brine (1  50 mL). 13. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 14. Purify the crude by flash column chromatography on silica using isocratic conditions of CH2Cl2/AcOEt/MeOH (90:8:2 (v/v/v)) (1.08 g cholesteryl tetraethylene glycol, 68%, global yield 50%). 3.4.2 Synthesis of Cholesteryl Tetraethylene Glycol Succinimidyl Carbonate

1. Dissolve cholesteryl tetraethylene glycol (1.08 g, 1.93 mmol, 1.0 equiv) in dry CH2Cl2 (25 mL), and cool the solution to 5  C. 2. Add phosgene solution ~20% in toluene (2.1 mL, 3.85 mmol, 2.0 equiv) and stir the mixture 2 h at 5  C (see Note 22). 3. Evaporate the crude to dryness and coevaporate two times with 20 mL of toluene. Use this intermediate without further purification. 4. Dissolve the crude in dry CH2Cl2 (20 mL) and cool the solution to 0  C. 5. Add N-hydroxysuccinimide (266 mg, 2.31 mmol, 1.2 equiv) dissolved in dry CH3CN (4 mL), and finally add DIEA (1.3 mL, 7.64 mmol, 4.0 equiv). 6. The mixture is allowed to warm from 0  C to RT while stirring for 1 h. 7. Evaporate the mixture to dryness. 8. Dissolve the crude in CH2Cl2 (30 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (1  40 mL). 9. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. Use this compound without further purification.

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1. Dissolve cholesteryl tetraethylene glycol succinimidyl carbonate (from Subheading 3.4.2; 1.36 g theor., 1.93 mmol, 1.4 equiv) and cyclo(RGDfK) peptide (12) (1.15 g, 1.38 mmol, 1.0 equiv) in dry DMF (40 mL). 2. Add DIEA (0.47 mL, 2.76 mmol, 2.0 equiv) and stir the mixture overnight (16 h) at RT. 3. Evaporate the mixture to dryness in a rotary evaporator. 4. Add 50 mL of CH3CN to the resulting crude and sonicate for 20 min. 5. Collect the precipitate by vacuum filtration using a glass funnel filter with sintered glass disc of 10–16 μm pore size. 6. Wash the precipitate with CH3CN (3  40 mL), H2O (3  40 mL), again with CH3CN (3  40 mL), and finally with Et2O (2  40 mL). 7. Dry the solid in a vacuum desiccator overnight. 8. Check the purity of the synthesized compound by HPLC and MS. Cholesteryl tetraethylene glycol-cyclo(RGDfK) conjugate (20) (1.33 g, 81%). HPLC purity 97% (210 nm) (see Note 23). ESI-MS (positive mode) m/z calcd. For C63H102N9O13 [M+H]+ 1192.8; found 1192.9.

3.5 Synthesis of HArg-Gly-Asp-Ser-OEGSH (21)

1. Add EDC·HCl (17.6 mg, 91.8 μmol, 1.5 equiv) and HOBt·H2O (14.1 mg, 92.0 mmol, 1.5 equiv) to a stirred solution of Boc-Arg(Pbf)-Gly-Asp(tBu)-Ser(tBu)-COOH peptide (9) (55.0 mg, 61.2 μmol, 1.0 equiv) in DMF (3 mL). 2. Add a solution of 1-(3-(Tritylthio)propanoyl-amino)-4,7,10trioxa-13-tridecanamine (6) (40.5 mg, 73.5 μmol, 1.2 equiv) in DMF (3 mL) and DIEA (22 μL, 122 μmol, 2.0 equiv). 3. Stir the resulting mixture overnight (16 h) at RT. 4. Remove DMF to dryness in a rotary evaporator. 5. Dissolve the crude in AcOEt (25 mL), pour it into a separation funnel, and wash the organic layer with saturated aqueous NaHCO3 (3  25 mL), 0.5 M HCl aqueous solution (2  25 mL), and brine (1  25 mL). 6. Dry the organic phase with MgSO4 and remove the solvent under reduced pressure. 7. Purify the crude by flash column chromatography on silica using CH2Cl2 and MeOH as solvents (0–5% MeOH in CH2Cl2) (73.8 mg of the fully protected peptide-OEG derivative, 84%). 8. Dissolve the product in 10 mL of a mixture of TFA/H2O/TIS (90:5:5, v/v/v), and leave to react for 2 h at RT (see Note 24).

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9. Concentrate the mixture in a rotary evaporator to reduce the volume to approximately one half of the total. 10. Add cold Et2O (seven- to eightfold with respect to the remaining volume of cleavage solution). 11. Centrifuge the precipitate for 5 min at 2200  g at 4  C. 12. Decant the ether and wash the precipitate two times with additional cold Et2O. 13. Dissolve the solid in H2O and lyophilize. 14. Dissolve the crude in a minimal volume of H2O and load into a 2 g Waters PoraPak™ Rxn column (see Note 6). 15. Elute the column with H2O (100 mL). 16. Elute with H2O:CH3CN (100 mL, 98:2 v/v, 0.1% TFA), with H2O:CH3CN (100 mL, 96:4 v/v, 0.1% TFA) and then with H2O:CH3CN (94:6 v/v, 0.1% TFA) until no product is detected by HPLC. 17. Monitor the collected fractions by HPLC and combine those containing the pure product. 18. Evaporate the CH3CN in a rotary evaporator and lyophilize the aqueous phase. 19. Check the purity of the synthesized compound by HPLC and MS. H-Arg-Gly-Asp-Ser-OEG-SH (21) (22 mg, 45%). HPLC purity 98% (210 nm) (see Note 25). ESI-MS (positive mode) m/z calcd. For C28H54N9O11S [M+H]+ 724.4; found 724.5.

4

Notes 1. Analytical RP-HPLC and mass spectra were performed on a Waters Alliance 2795 with an automated injector and a photodiode array detector Waters 2996 coupled to an electrospray ion source (ESI-MS) Micromass ZQ mass detector, using the MassLynx 4.1 software. The reversed-phase analytical columns used were: XSelect C18 (4.6 mm  50 mm, 3.5 μm), XBridge BEH C18 (4.6 mm  100 mm, 3.5 μm), SunFire C18 (4.6 mm  100 mm, 3.5 μm), and Symmetry300 C4 (4.6 mm  250 mm, 5 μm). 2. The standard mass analyses were performed in the HPLC-MS system described in Note 1. High-resolution mass spectroscopy (HRMS) was carried out using an LC/MSD-TOF spectrometer from Agilent Technologies. 3. Imidazole-1-sulfonyl azide hydrochloride is commercially available from several sources; however, it can be easily prepared on a large scale from inexpensive materials in a one-pot reaction [26].

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4. The solid formed corresponds to the triphenylphosphine oxide (PPh3O). Although the major part is removed in the first filtration, more solid appears while evaporating the filtrate. After the reduction of the volume to approximately one half of the total, a second filtration can be carried out; however, it is not strictly necessary because the remaining excess of PPh3O is completely and easily removed in the column chromatography. 5. In this reaction conditions, with a highly saturated aqueous phase, two phases can be distinguished; thus it is recommended a vigorous stirring for the appropriate progression of the reaction. 6. Before the loading of the product, it is recommended to wash the column with CH3CN (3–4 column volumes) and then with H2O (4–5 column volumes to completely remove the CH3CN). 7. It is important to maintain the basicity of the mixture. The pH slightly decreases during the reaction, and it is recommended a second addition of base to force the reaction to completion. 8. It is important to ensure that 3-(Tritylthio)propionic acid is totally consumed (monitor by HPLC), and thus a little excess of the OEG reagent is used. If no 3-(Tritylthio)propionic acid remains in the crude when the reaction is finished, the desired compound can be easily isolated by aqueous work-up procedures. Otherwise, column chromatography is needed to obtain the pure product. 9. The use of Zn/NH4Cl mixture is an alternative method to reduce the azido group to amine. However, this step can also be carried out by a classical Pd/C catalyzed hydrogenation. 10. PyBOP can be used as coupling reagent as a replacement for HBTU. In terms of coupling efficacy, both reagents proceed similarly with excellent yields. However, both reagents show minor drawbacks from the point of view of the by-products. On one hand, the tris(pyrrolidine-1-yl)phosphine oxide formed in PyBOP-mediated couplings is difficult to remove completely by aqueous work-up procedures. On the other hand, the HBTU can also react with the free amine of the OEG and form the corresponding tetramethyluronium derivative, thus reducing the equivalents of reagent available for the coupling reaction. 11. The work-up procedures are similar both in the case of using PyBOP or HBTU coupling reagents, but there is a difference in the aqueous acidic extraction. If PyBOP is used, washings with a citric acid solution are enough to remove the excess of OEG reagent. However, when HBTU is used, the excess of OEG reagent is in the form of the tetramethyluronium

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derivative, and it is not extracted to the aqueous phase. In this case it is necessary to use a 1 M HCl aqueous solution to completely remove this by-product. 12. Steps 9–11 are performed to remove the tris(pyrrolidine-1-yl) phosphine oxide formed in PyBOP-mediated couplings. However, these steps are not required when the coupling reaction is performed with HBTU. 13. It is important to ensure a complete elimination of the MeOH. Traces of this solvent may be retained in products of this nature, and, occasionally, in a further step of acylation of the free carboxylic acids, significant amounts of the undesired methyl ester derivative have been detected. To avoid this issue, it is recommended to carry out two to three coevaporations with acetonitrile previous to acylation reactions. Another option is to replace MeOH with 2-propanol in the hydrogenation reaction, which also works well, although more slowly. 14. The final mixture should not exceed the ratio of 10 mL solvent/g resin. If the mixture is not completely soluble, add a small amount of DMF. 15. A recommended protocol to dry a resin is firstly carry out washes with DCM (3  1 min) and MeOH (3  1 min), then dry by vacuum for 10–15 min, and finally dry the resin overnight in a vacuum desiccator at RT. 16. OxymaPure was developed as an efficient additive for peptide synthesis, becoming a safer alternative reagent to HOBt and HOAt, whose potential risk of explosion has been reported. Excellent coupling efficiency and very low degrees of epimerization are achieved when using OxymaPure. It has been described that the performance of this reagent is superior to that of HOBt and comparable to that of HOAt [27]. 17. The preactivation of 1–2 min after the DIC addition is not carried out when arginine is the amino acid to be coupled, in order to minimize the side reaction of delta-lactam formation that occurs with this residue. In this case, it is recommended to add the solution of the Fmoc-Arg(Pbf)-OH and Oxyma to the resin and, finally, the carbodiimide. 18. Place a few beads of the resin into a glass tube and add six drops of Kaiser test reagent A and two drops of Kaiser test reagent B. Mix well and heat the tube to 110  C for 3 min. Yellow color is a negative result, indicating no presence of free amines and, thus, a complete coupling. Blue or slightly blue color indicates a positive result; it is the presence of unreacted primary amino groups, and consequently the coupling has to be repeated. 19. Some protocols recommend to directly collect the TFA-CH2Cl2 filtrates and concentrate the mixture either in a rotary evaporator or under a stream of nitrogen. In these

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conditions, since the CH2Cl2 is more volatile, the concentration of TFA in the mixture increases during the evaporation, and this could cause the undesired partial loss of some protecting groups of the peptide. (Actually, this problem was detected in one of the first attempts to synthesize peptide 9, with a partial loss of tBu group of the aspartic acid residue.) Thus, we strongly suggest following the described procedure in this protocol. 20. This protocol renders the desired peptide as the bis(trifluoroacetate) salt with purities >90–95% without further purification. 21. The HPLC analysis was carried out using the SunFire C18 column (see Note 1) and a linear gradient of 0–100% CH3CN (0.036% TFA) in H2O (0.045% TFA) over 8 min at a flow rate of 1 mL/min. 22. The progression of the reaction is monitored by TLC (hexane/ AcOEt 35:65 (v/v), KMnO4 stains) to ensure the total consumption of the starting material. Otherwise, add extra phosgene solution to guarantee the complete formation of the intermediate cholesteryl tetraethylene glycol chloroformate. 23. The HPLC analysis was carried out using the Symmetry C4 column (see Note 1) and a linear gradient of 5–100% CH3CN (0.07% HCOOH) in H2O (0.1% HCOOH) over 30 min at a flow rate of 1 mL/min. 24. The mixture used to remove the protecting groups is very similar to cleavage solution A, but it contains a higher percentage of scavengers. It has been detected partial tert-butylation of the thiol group when using cleavage solution A, even with the use of 1% of 1,2-ethanedithiol as scavenger. To minimize the amount of side product, one strategy is to increase the amount of H2O and TIS and work at high dilution. In these conditions the tert-butylated product is reduced to 5–7%. 25. The HPLC analysis was carried out using the XBridge BEH C18 column (see Note 1) and a linear gradient of 0–30% CH3CN (0.036% TFA) in H2O (0.045% TFA) over 8 min at a flow rate of 1 mL/min. References 1. Zelzer M, Ulijn RV (2010) Next-generation peptide nanomaterials: molecular networks, interfaces and supramolecular functionality. Chem Soc Rev 39:3351–3357 2. Rong L, Qin SY, Zhang C, Cheng YJ, Feng J, Wang SB, Zhang XZ (2018) Biomedical applications of functional peptides in nano-systems. Mater Today Chem 9:91–102

3. Sun L, Zheng C, Webster TJ (2017) Selfassembled peptide nanomaterials for biomedical applications: promises and pitfalls. Int J Nanomedicine 12:73–86 4. Sun H, Dong Y, Feijen J, Zhong Z (2018) Peptide-decorated polymeric nanomedicines for precision cancer therapy. J Control Release 290:11–27

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5. Henninot A, Collins JC, Nuss JM (2018) The current state of peptide drug discovery: back to the future? J Med Chem 61:1382–1414 6. Al Musaimi O, Al Shaer D, De la Torre BG, Albericio F (2018) 2017 FDA peptide harvest. Pharmaceuticals 11:1–10 7. Zhang C, Wu W, Li RQ, Qiu WX, Zhuang ZN, Cheng SX, Zhang XZ (2018) Peptide-based multifunctional nanomaterials for tumor imaging and therapy. Adv Funct Mater 28:1–22 8. Richard J (2017) Challenges in oral peptide delivery: lessons learnt from the clinic and future prospects. Ther Deliv 8:663–684 9. Pasut G, Veronese FM (2012) State of the art in PEGylation: the great versatility achieved after forty years of research. J Control Release 161:461–472 10. Turecek PL, Bossard MJ, Schoetens F, Ivens IA (2016) PEGylation of biopharmaceuticals: a review of chemistry and nonclinical safety information of approved drugs. J Pharm Sci 105:460–475 11. Mammen M, Choi SK, Whitesides GM (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed Engl 37:2754–2794 12. Mehta D, Leong N, McLeod VM, Kelly BD, Pathak R, Owen DJ, Porter CJH, Kaminskas LM (2018) Reducing dendrimer generation and PEG chain length increases drug release and promotes anticancer activity of PEGylated polylysine dendrimers conjugated with doxorubicin via a cathepsin-cleavable peptide linker. Mol Pharm 15:4568–4576 13. Da˚nmark S, Aronsson C, Aili D (2016) Tailoring supramolecular peptide-poly(ethylene glycol) hydrogels by coiled coil self-assembly and self-sorting. Biomacromolecules 17:2260–2267 14. Huang YQ, Yuan JD, Ding HF, Song YS, Qian G, Wang JL, Ji M, Zhang Y (2018) Design, synthesis and pharmacological evaluation of a novel PEG-cRGD-conjugated irinotecan derivative as potential antitumor agent. Eur J Med Chem 158:82–90 15. Dai Z, Yao Q, Zhu L (2016) MMP2-sensitive PEG-lipid copolymers: a new type of tumortargeted P-glycoprotein inhibitor. ACS Appl Mater Interfaces 8:12661–12673 16. Chen C, Duan Z, Yuan Y, Li R, Pang L, Liang J, Xu X, Wang J (2017) Peptide-22 and cyclic RGD functionalized liposomes for glioma targeting drug delivery overcoming BBB and BBTB. ACS Appl Mater Interfaces 9:5864–5873

17. Liu HJ, Luan X, Feng HY, Dong X, Yang SC, Chen ZJ, Cai QY, Lu Q, Zhang Y, Sun P, Zhao M, Chen HZ, Lovell JF, Fang C (2018) Integrated combination treatment using a “smart” chemotherapy and microRNA delivery system improves outcomes in an orthotopic colorectal cancer model. Adv Funct Mater 28:1–15 18. Pulido D, Albericio F, Royo M (2014) Controlling multivalency and multimodality: up to pentamodal dendritic platforms based on diethylenetriaminepentaacetic acid cores. Org Lett 16:1318–1321 19. Simo´n-Gracia L, Pulido D, Sevrin C, Grandfils C, Albericio F, Royo M (2013) Biocompatible, multifunctional, and well-defined OEG-based dendritic platforms for biomedical applications. Org Biomol Chem 11:4109–4121 20. Fransen P, Pulido D, Sevrin C, Grandfils C, Albericio F, Royo M (2014) High control, fast growth OEG-based dendron synthesis via a sequential two-step process of copper-free diazo transfer and click chemistry. Macromolecules 47:2585–2591 21. Seelbach RJ, Fransen P, Peroglio M, Pulido D, Lopez-Chicon P, Duttehhoefer F, Sauerbier S, Freiman T, Niemeyer P, Semino C, Albericio F, Alini M, Royo M, Mata A, Eglin D (2014) Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels. Acta Biomater 10:4340–4350 22. Seelbach RJ, Fransen P, Pulido D, D’Este M, Duttenhoefer F, Sauerbier S, Freiman T, Niemeyer P, Albericio F, Alini M, Royo M, Mata A, Eglin D (2015) Injectable hyaluronan hydrogels with peptide-binding dendrimers modulate the controlled release of BMP-2 and TGF-β1. Macromol Biosci 15:1035–1044 23. Fransen P, Pulido D, Simo´n-Gracia L, Candiota AP, Aru´s C, Albericio F, Royo M (2015) r1 and r2 relaxivities of dendrons based on a OEG-DTPA architecture: effect of Gd3+ placement and dendron functionalization. J Nanotechnol 2015:1–8 24. Cabrera I, Elizondo E, Esteban O, Corchero JL, Melgarejo M, Pulido D, Co´rdoba A, Moreno E, Unzueta U, Vazquez E, Abasolo I, Schwartz S Jr, Villaverde A, Albericio F, Royo M, Garcı´a-Parajo MF, Ventosa N, Veciana J (2013) Multifunctional nanovesicle-bioactive conjugates prepared by a one-step scalable method using CO2-expanded solvents. Nano Lett 13:3766–3774

Peptide-OEG Conjugates for Multivalent Nanomaterials 25. Cabrera I, Abasolo I, Corchero JL, Elizondo E, Gil PR, Moreno E, Faraudo J, Sala S, Bueno D, Gonza´lez-Mira E, Rivas M, Melgarejo M, Pulido D, Albericio F, Royo M, Villaverde A, Garcı´a-Parajo M, Schwartz S Jr, Ventosa N, Veciana J (2016) α-Galactosidase-A loadednanoliposomes with enhanced enzymatic activity and intracellular penetration. Adv Healthcare Mater 5:829–840

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Chapter 8 Phage Display Methodologies Agathe Urvoas, Philippe Minard, and Patrice Soumillion Abstract In vitro selection of bacteriophages displaying specific protein binders from large combinatorial libraries is a well-established and very powerful technology. Therapeutic antibodies that have been evolved by phage display are now on the market and various non-antibody scaffolds are currently being developed as the nextgeneration competitors. In this chapter, after presenting the many possible proteins that can be engineered by phage display, we describe some modern methods for generating highly diverse phage libraries as well as selection protocols and experimental tips that will help researchers implementing the technology in their laboratory. Key words Phage display, In vitro selection, Library, Binder, Artificial protein, Antibody

1

Introduction Phage display is a powerful technology for engineering tailor-made protein binders. Since its first description in 1985 [1], several thousands of research articles reporting applications of this technology have been published. In 2018, half of the Nobel Prize in Chemistry has been awarded to George Smith, who first applied phage display to peptides and Gregory Winter who first applied phage display to engineer antibodies. As the result of a simple genetic fusion between a chosen open reading frame (ORF) and a gene encoding a filamentous phage coat protein, the corresponding peptide, protein, or antibody fragment can be expressed at the surface of a phage particle, physically linked to its genetic information via the coat. Using large libraries of variants, the selective capture of a phage based on its specific interaction with an immobilized ligand allows the concomitant selection of the genetic information, which can be amplified by simple phage infection of bacteria (Fig. 1). The selection process is conducted entirely in vitro, in defined and controlled conditions. This chapter focuses on phage display for protein engineering and will not describe other applications such as peptide or whole

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_8, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Immobilized target

gs

hin Was

Next round Sequence

Elution

Infection and amplification

Fig. 1 The principle of phage display is based on in vitro selection of peptides or proteins expressed at the surface of filamentous phages. In a “bio-panning” experiment, specific binders can be captured from a library using an immobilized target ligand and their genes amplified by simple phage infection. After several rounds of bio-panning, the phage population is enriched into specific binders. Each phage encapsulates the gene encoding the protein or peptide expressed on its surface, allowing recovering the sequence of the selected binders

phage engineering neither all the alternative display methods that have been developed to circumvent some of the limitations of the phage technology [2]. Several interesting reviews and books should also be of interest for scientists that are using or planning to use phage display [3–12]. Before entering into practical considerations, our introduction is focused on the large diversity of proteins that have been engineered by phage display (Fig. 2). 1.1 Selecting a Protein Scaffold 1.1.1 Antibody Scaffold

Fab and scFV

One of the most successful applications of the phage display technology is the selection of antibody-derived proteins featuring tight binding for chosen molecular or cellular targets. Nowadays, engineered therapeutic antibodies in multiple formats have become leading products for novel therapeutic approaches, notably for cancer treatments [13, 14]. Immunotherapy is also promising for the treatment of other pathologies related to inflammation or neuronal diseases [15]. Because of their large size, hetero-tetrameric structure, and multiple disulfide bonds, whole antibodies are difficult to handle as recombinant proteins and are inefficiently expressed and displayed on phages. Dimeric Fab and monomeric scFv fragments have thus been used instead [5, 8, 10]. Fab fragments are more difficult to handle but tend to be more stable than scFvs and are easier to

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Fig. 2 Representative structure of alternative protein scaffolds either isolated (top row) or in complex with a cognate protein target (bottom row). The evolved protein binder is shown in orange and the target in purple. From left to right (PDB code) (a) affibody and complex with HER2 (3MZW); (b) monobody and complex with F
/ H+ antiporter (6D0J); (c) Anticalin and complex with VEGF (4qaf); (d) Darpin and complex with Caspase 2 (2P2C); (e) αRep and complex with α Tubulin (6GWD); (f) Affimer and complex with Fcg receptor (5ML9)

convert into full-length IgG for further applications. ScFv fragments are easier to handle for recombinant expression in E. coli but, depending on the size of the linker, can be subject to oligomerization by domain swapping. Such multimers with an increased avidity for their targets (cancer cells, viruses) and a longer half-life in vivo may, however, be interesting for some applications. Immune, naı¨ve, or synthetic libraries of Fab or scFv can be prepared (for review [16–18]) starting from V genes of immunized or nonimmunized organisms, or from artificial DNA assembly, respectively. Immune libraries, from antigen-injected animals or microbe-infected humans, present a bias of specificity for the antigen or microbe and are therefore dedicated to engineering binders against a single target. The construction of large naı¨ve and synthetic libraries (109–1010 clones) were developed as a more versatile approach, allowing the selection of binders with nanomolar affinities for almost any protein targets [4, 19]. Single Domain VHH

A specific class of antibodies found in Camelidae contains only two heavy chains but no light chains. The variable region (VHH) of these antibodies is the smallest antigen-binding fragment (11–15 kDa). Featuring low complexity of the binding site (only three hypervariable loops), high production levels in prokaryotic and eukaryotic hosts, and good stabilities, these engineered VHH

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scaffolds, named nanobodies, are clearly an interesting alternative to classic antibody derivatives [20]. The phage selection of targetspecific nanobodies usually relies on an immunization of an appropriate animal (e.g., lama, alpaga, dromedary) followed by the construction of VHH gene library amplified from its peripheral blood. Only recently, efficient synthetic universal VHH libraries have been described [21–23] and are of sufficient quality to allow the selection of high-affinity nanobodies for any protein target. Therapeutic nanobodies are being developed, and several clinical trials are currently ongoing [24]. 1.1.2 Alternative Scaffolds

In addition to the disadvantages mentioned above, going from low expression of large hetero-oligomeric antibodies to misfolding or domain swapping of artificially fused protein domains, most antibody-based domains are not able to fold in the reducing environment of the cytoplasm of living cell, including VHH-derived nanobodies that also have a stabilizing disulfide bond. This limits any potential usage of antibody-derived binders as effector/modulator of biological function within living cells. To overcome these limitations as well as patent-related restrictions, researchers turned to other proteins for engineering binders. A set of recent reviews have listed more than 40 different proteins proposed as possible scaffolds, at various stages of developments [25–28]. These were selected generally as small monomeric proteins, with favorable expression properties. Most of proposed scaffolds are single-domain and disulfide-free proteins, but proteins made by concatenation of single domains have also resulted in tight binders [29, 30]. As the starting protein is subjected to extensive sequence modification, which might have destabilizing effect, a high initial stability may be an important criterion [31]. For therapeutic applications, some scaffolds of human origin have been selected such as to minimize potential immunogenicity. Enzymes have also been used as scaffolds for engineering binding sites at the vicinity of the active site giving rise to allosteric regulation properties [32, 33]. Most proposed protein scaffolds have been described in the past two decades and still represent a large field of development for the future. For a limited subset of them, highly diverse libraries have been described from which, tight and specific binders against a range of different targets were selected and effectively produced. Some of these alternative scaffolds have indeed been patented and are now commercially developed in new biotech companies, namely Affibody® (Affibody), Affimer® (Avacta), Anticalin® (Pieris Pharmaceuticals), AdnectinTM (BMS), DARPin® (Molecular Partners), Kunitz Domain (Shire), and Nanofitin® (Affilogic) [34]. Among the limitless biotechnological applications of these new type of protein binders, therapeutic proteins are also developed, with some of them already in clinical trials [35, 36].

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Monodomain Proteins

Affibodies are disulfide-free small helical proteins derived from a bacterial protein domain similar to Protein A. Side chains located on the outside surface of the three helix bundles have been randomized to create large phage display libraries [37]. Structural data have been described, which explain how this small and partially folded architecture can adapt to a range of binding surfaces and fold upon binding to its target [38, 39]. The small size of affibodies results in a fast clearance and good penetration in solid tumors [40]. Affibodies are developed for a wide range of applications including microarrays [29] and specific binding reagents for affinity chromatography [41]. Fibronectin III domains have the characteristic fold of immunoglobulin domains but without disulfide bonds. Several binders were selected from libraries of fibronectin variants for which two or three pseudo-CDR loops were fully or partially randomized with only serine or tyrosine [42, 43], a strategy that was previously applied for antibodies [44]. Lipocalins are a family of proteins found in many organisms, including humans, that naturally bind specifically small hydrophobic molecules. These proteins have a classical up and down β barrel topology and bind specific ligands in a funnel-shaped binding site located in the center of the β stands on one end of the barrel. By randomizing the side chains forming this cavity, variants, called anticalins, were initially engineered to bind small molecule targets [45]. Interestingly, by randomizing inter-strand loops, the same scaffold family has been also successfully used to engineer tight protein binders [46]. The lipocalin family also includes a human protein (tear lipocalin) that can be used to minimize immunological response in medical applications. Several anticalins are now in clinical development [47]. The Affimer scaffold derives from both the human stefin A protein [48] and plant cystatin protein [49]. It is composed of four antiparallel β-sheet strands flanking a central α-helix. Affimer libraries were generated by randomizing the inter-strand loops and screened by phage display against a broad range of targets including a small organic molecule. Various promising developments of affimer-derived binders for biochemical and cellular applications have been described including tools for immune-like affinity assays, dissection of intracellular signaling pathways, modulation of extracellular receptor and ion channel functions, or in vivo imaging [50].

Repeat Proteins as Modular Scaffolds

Repeat proteins present an extended or closed folded structure resulting from the concatenation of small structural motifs (20–40 residues) [51]. Several families of repeat proteins are currently explored as very promising alternative scaffolds candidates. Although diverse, these protein families have all been extensively

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used by natural evolution to make a wide diversity of very tight binding proteins. This remarkable evolutionary success directly results from their structure made by juxtaposition of selfcompatible modules. Within each small module, structurally important residues involved in intra- and inter-module stability can be identified from consensus analysis [52]. The remaining residues are highly variable between modules and often exposed to the solvent. In the folded protein, the juxtaposition of modules with variable outside surfaces creates potentially large and chemically diverse interaction surfaces. This modular organization has opened new routes for combinatorial engineering of artificial repeat proteins. Besides combinatorial methods for generating libraries, modules can also be combined for affinity maturation or for multifunctional binding. The pioneering DARPins, developed by Plu¨ckthun’s group, are derived from ankyrin repeat proteins and have been particularly successful to generate very tight binding proteins against a number of targets [53]. Some of these proteins have been demonstrated to act as specific intracellular inhibitors [54], or as scaffolding components in co-crystallization experiments with soluble proteins and membrane proteins [55]. Although DARPins were initially selected by ribosome display, tight binders were also selected by phage display [56]. Several therapeutic candidates based on the DARPin® technology are currently developed by Molecular Partners, some of them have reached phase II or III in clinical trials. Another scaffold based on leucine-rich repeat proteins (LRR) originates from a primordial adaptive immune system found in jawless vertebrates. The LRR hypervariable regions can be diversified by recombining gene fragments, like in classical immune systems [57]. The group of Kim has recently described a synthetic phage library of LRR proteins based on these natural repeat proteins, named Repebodies, and binders were selected against a series of protein targets [58, 59]. A last family of artificial repeat proteins, named αRep, was designed from the concatenation of structural HEAT-like helical thermostable repeats [60]. Phage libraries presenting a dual diversity were generated by varying the number of repeats and randomizing six hypervariable positions in each repeat. The structures of αReps look like curved solenoids of various lengths with the randomized surface of interaction located on the concave side. αReps are easily produced in E. coli in large amounts (>30 mg/L), highly stable (Tm > 70  C) and disulfide-free. From a highly diverse phage library (109 variants), binders with high selectivity and affinity for a variety of protein targets (KD from nM to μM) were selected [61]. Various αRep-based applications have been explored [62]. In structural biology, they have proven to be efficient co-crystallization chaperones, as shown with ten reported examples of αRep/target complex X-ray structures [63–65]. They can be

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functionally expressed in living eukaryotic cells and could therefore be used for the development of intracellular tracers [66]. The design of artificial metalloenzymes by coupling organometallic complexes into an αRep protein scaffold [67] and the development of generic biosensors [68] and nanomaterial objects [69, 70] are also in progress. 1.2 Important Practical Considerations

When starting a phage display project, several choices have to be made besides the protein to be engineered and before building a library and performing affinity selections. In this section, we describe the main features and alternatives with their respective advantages and disadvantages. Those are also summarized in Table 1. In the morphogenesis of the filamentous phage [7], coat proteins are initially targeted to the inner membrane of E. coli prior to their assembly into the phage particle. The protein to be displayed must reside in the periplasmic space anchored to one of the coat proteins. Hence, it will necessitate the cloning of its gene between a sequence encoding a signal peptide for export and a sequence encoding a full or truncated coat protein. Although each of the five coat proteins of the filamentous phage has been used in phage display, a pIII-fusion is generally chosen for the surface exposure of a protein. Either phage or phagemid vectors can be used, each of which with their own advantages and disadvantages. In the case of phage vectors, the gene of the recombinant protein is included in the phage genome, fused to the full pIII gene and under its native weak and noninducible promoter. Although phage particles should display only fusion proteins, full display remains an exception as in vivo proteolysis will often partially remove the fusion protein from the pIII. Typical display levels ranging between 0.2 and 3 proteins per phage particle are generally obtained. Polyvalent phages can afford the selection of weak binders by avidity effects if the density of immobilized ligand is high. Phage vectors also contain antibiotic resistance markers allowing the detection of infected bacteria as colonies. Regarding disadvantages, all the cloning and DNA manipulation must be performed using the replicative form (RF) of the phage, which is more difficult to produce in large amounts and high purity. For some foreign genes, genetic instability is also sometimes evoked. In the case of phagemid vectors, the recombinant protein is encoded as a fusion protein with pIII or truncated pIII. The other proteins required to make a functional phage particle are provided by a helper phage. Phagemids have all the advantages of plasmids with regard to cloning, DNA manipulation, control of expression with promoters, and genetic stability. However, common inducible promoters such as pLac are stronger than the endogenous pIII promoter, and their use could result in toxicity effects. Soluble proteins can be produced without subcloning if an amber codon is inserted between

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Table 1 Key practical features in setting up a phage display system Key feature

Pros

Cons

Vector Phage

Large vector (around 10 kbp); library Natural context; antibiotic resistance of construction is more laborious; no infected colonies; no need for helper transcriptional control phage; high display level (monovalent to polyvalent)

Phagemida

Small vector (¼ plasmid); library construction is easy; transcriptional control; monovalent display

Need for helper phage (brings its own coat proteins); low level of display

Coat protein for display pIIIa

High accessibility, fusion with N-terminal C-terminal not accessible, infectivity of pIII, 0–5 displayed proteins per phage decrease if polyvalent display

Truncated pIII (CT-pIII)a

Smaller size, might be more appropriate for Require the expression of a wt pIII displaying large proteins

pVIII

High level of display (up to thousands per Display of peptides only phage)

pVI

Fusion with C-terminal of pVI allowed

pVII, pIX

Display on the other side of the filament Very few examples (possible combination with pIII display)

Very few examples

Promoter pPIII (endogeneous phage promoter of pIII)

Naturally adapted to phage infection cycle Not controllable

pLac (IPTGinducible)

Widely used, controllable

Leaky (unless LacIq genotype)

pPsp (phage shock promoter)

Display of toxic proteins, induced by helper phage infection

Few examples

Hybrid pLac-pT7 Used for both display (with pLac in supE strains) and soluble expression (with pT7 in DE3 strains), controllable

Requires stop codon between protein gene and pIII gene

Linker AAIEGRAAa GGGSGGGS

a

Cleavable with factor Xa or trypsin

Susceptible to in vivo proteolysis

Resistant to in vivo proteolysis

Non-cleavable (continued)

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Table 1 (continued) Key feature

Pros

Cons

SecB-dependent (PelB)a

Widely used, no toxicity, convenient for most naturally exported proteins

Not compatible with many cytosolic proteins and rapidly folding proteins

SRP-dependent (DsbA)a

Required for rapidly folding and stable proteins

Expression associated with a fitness cost

TAT-dependent

Potentially useful for cofactor-bound proteins

Few examples, incompatible with linkers or unfolded regions

Signal sequence for export

a

Most commonly used

the gene encoding the displayed protein and the pIII gene. Phagemid vectors can then become very efficient expression vectors of selected proteins if a powerful T7-based promoter is included in addition to the classical weaker promoters used for display on the phage surface [71]. This type of vectors, although not useful when working with antibodies or poor folding proteins, are extremely convenient for highly expressed scaffold proteins. An interesting promoter for the display of toxic proteins using phagemid vectors is the phage shock promoter (pPsp) since the expression of the fusion protein will be induced only when the morphogenesis of phage particles is triggered, i.e., upon infection with the helper phage [72]. A disadvantage of phagemids is the relatively low level of surface display, which results generally in a large proportion of phage particles that are not displaying any fusion protein. This is due to the competition between the fusion protein and the wildtype pIII coming from the helper phage. Selection cycles are also longer because of the need of superinfection with the helper phage. Several strategies were attempted in order to combine the advantages of the phagemid and phage libraries, by creating helper phages mutated or deleted in gene 3 (hyperphage), or packaging bacterial cell lines; in particular; those strategies were described for improving the display level of antibody fragments and the subsequent selection efficiency [73–76]. The choice of an adequate signal sequence is also critical for successful display of some proteins. In E. coli, three different types of signal sequences exist. SecB-dependent sequences promote posttranslational translocation and may not be suitable if proteins are very stable and rapidly folded in the cytoplasm. SRP-dependent sequences promote co-translational translocation so that the protein will not “see” the cytoplasm and will fold directly in the periplasm [56]. However, the use of such signal sequences is associated with a fitness cost that may result in poor genetic stability.

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Finally, TAT-based signal sequences promote post-folding translocation and, in principle, may be used for displaying cofactorcontaining proteins. TAT pathway could also be used to display stable proteins and could be used to screen folded protein [77]. It has been shown that the TAT pathway-associated chaperones retain in the cytoplasm and prevent export of proteins that include long linkers and nonfolded sequences. The pIII proteins often used for phage display do have such a long linker sequence between the N terminal N1 and N2 domains and the C terminal domains. For an efficient display based on a TAT pathway export system, it is therefore critical to use a truncated pIII as display construct, i.e., a pIII without the N domains and the linker sequences [78]. With pIII display, the carboxy-terminus of the protein of interest is fused to the amino-terminus of the coat protein via a peptide linker. This linker must be of sufficient length for allowing proper folding of both proteins. Moreover, in some cases, a linker which is cleavable by a specific endoprotease may be interesting, since it can afford specific phage elution from the selecting support or can also be used for increasing the phage infectivity, mainly in case of polyvalent display. Finally, the linker should not be too susceptible to in vivo proteolysis for avoiding the cleavage of the fusion protein during phage production. AAIEGRAA is a convenient cleavable linker by Factor Xa endoprotease or trypsin, and quite resistant to in vivo proteolysis; GGGSGGGS is a noncleavable, flexible, and hydrophilic linker, highly resistant to in vivo proteolysis. Monovalent display of N-terminal-anchored proteins using phage vectors has also been achieved through linker engineering of pIII fusion [79].

2

Materials Filamentous phages F1, fd, or M13 only infect Escherichia coli strain harboring the F0 episome. Classical strains used in phage display are TG1, XL1 Blue, and JM109. Growth media: LB: tryptone (10 g), yeast extract (5 g), and NaCl (5 g) for 1 L of sterilized medium (autoclave). 2YT: tryptone (16 g), yeast extract (10 g), and NaCl (5 g) for 1 L of medium. Dissolve by boiling and stirring and adjust pH to 7 with NaOH 5 N before autoclaving. Buffers (sterilized by autoclaving): TE: 10 mM Tris–Cl, 1 mM EDTA, pH 8.0. TBS: 50 mM Tris–Cl, 150 mM NaCl, pH 7.6. TBST: 50 mM Tris–Cl, 150 mM NaCl, 0.05% Tween 20, pH 7.6. PBS: 10 mM Phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4.

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Short oligonucleotides were ordered as desalted grade (selective precipitation optimized process), while long degenerate ones were purified by polyacrylamide electrophoresis. Phusion DNA polymerase, DpnI, T5 exonuclease, and Taq DNA ligase were purchased from New England Biolabs.

3

Methods In this section, we describe well-tested methods from the building of a library to the affinity capture selections and downstream screening. We focused on protocols developed with commonly used phagemid-based libraries. Conventionally, the terms phagemid and phage will be used to designate the double-stranded DNA form and the filamentous particle, respectively, even though a particle containing a phagemid-DNA is not really a phage in the biological sense (it is a transducing particle and not an infective one). We also add some notes for those who might be interested in working with non-phagemid phage libraries.

3.1 Constructing a Library of DNA In Vitro

Building a highly diverse and high-quality library is the key to the success of a phage display project. In most cases, libraries are constructed by classical random mutagenesis methods such as error prone PCR, DNA shuffling and incorporation of degenerate oligonucleotides, or by recovery of natural repertoires of antibodies. Many protocols can be found in the literature. Diversity in oligonucleotide-based libraries was originally often introduced by complete random substitution of selected positions using NNK, NNS, or NNB degenerate codons, where all 20 amino acids and one stop codon are encoded by the degenerate codons. However, this strategy suffers from serious drawbacks: the proportion of substituting side chains cannot be adjusted, and consequently a fraction of sequences are severely destabilized for example by the presence of prolines in α-helices, or by large side chains in positions leading to steric clashes. Partially degenerate codons can be used to minimize the problem. For example, VRN or NTN degenerate codons will encode respectively nine hydrophilic or five hydrophobic residues. Spiked codons can also be used for favoring a specific residue at a specific position while enabling its mutation in a controlled fraction of the library. For example, an AAT codon contaminated with 10% B in the first and second positions will encode around 84% of Asn residue and 16% of the 19 others. Libraries featuring highly simplified side chain diversity, with only three amino acids (Y, G, S) in randomized positions, also proved to be remarkably efficient [80, 81]. Nowadays, oligonucleotide and gene synthesis methods based on trinucleotide

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precursors, or by massively parallel oligonucleotide synthesis, allow to control the proportion of codons and to precisely tune the introduced diversity at each position on the basis of structural and/or evolutive data. In every protocol, the phagemid library is prepared in vitro, and the efficiency of the last transformation step will determine the diversity which should be as high as possible, meaning that the efficiencies of all the preceding steps must be high (see Note 1 for practical tricks). Besides classical methodologies based on PCR, restriction and ligation, progress in molecular biology and DNA synthesis capabilities are opening new routes for library building. Massive parallel synthesis of oligonucleotides affords the assembly of libraries with exquisite control on the content. Avoiding the use of restriction enzymes such as with the Gibson assembly method is also facilitating the design of the library (see Chapter on chromosomal libraries for detailed protocols). Here, we describe two specific methodologies that are less known but have been used to build high-quality and high-diversity libraries. 3.1.1 QuickLib for Rapid Cloning of Degenerate Oligonucleotides [82]

The method is illustrated in Fig. 3. It is based on the PCR-amplification of full phagemid vector using a pair of primers designed with a degenerate sequence and short overlapping ends. The linear PCR product is then simply recircularized using a Gibson assembly mix, thanks to annealing ends. Short overlapping ends (20–30 nt) are sufficient since the assembly reaction is intramolecular. DpnI is also added to the Gibson mix in order to remove all starting material (methylated DNA). The DNA product is then ready for transformation. (a) The quality of the phagemid matrix is the determinant for efficient full-length PCR amplification. Freshly prepared DNA was used in all the described experiments, avoiding, if possible, freezing/thawing cycles. The synthesis of plasmid libraries was performed in 50 μl total volume of mix containing the following: 1 Go-Taq polymerase Flexi Buffer from Promega (no matter which polymerase was used), 4 mM of MgCl2, 0.5 mM of each dNTPs, 0.5 μM of the forward degenerate primer, 1 μM of the reverse primer, 75 pM of the vector matrix, and 1 U of Phusion DNA polymerase. The following PCR protocol was applied: initial denaturation at 95  C for 4 min, followed by 20 cycles of denaturation (95  C, 90 s), annealing (55  C, 30 s) and elongation (72  C, 7 min), and ending by a final elongation (72  C, 10 min). The number of cycles was reduced to 15 for bigger plasmids (>8 kb). In general, the number of cycles should not be pushed too high to avoid decreasing the dNTPs/primers concentrations to the point where self-priming of the combinatorial library sequences becomes significant and lead to DNA aggregates.

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a

PCR

e

b

DpnI T5 exonuclease Phusion DNA pol Taq DNA ligase

c

d

Fig. 3 Scheme of the QuickLib method for cloning degenerate oligonucleotides into a phagemid. (a) The entire phagemid vector is initially PCR-amplified using an asymmetric pair of primers sharing complementary 50 ends (dark blue), one of them comprises a central degenerate region. (b) The library of linearized synthetic plasmids is then digested by T5 exonuclease, (c) affording annealing of complementary ends. (d) The subsequent action of a DNA polymerase and ligase result in circularization of the plasmid library. (e) The methylated starting vector is selectively eliminated by DpnI treatment

(b) On ice, 50 μl of crude PCR product (around 5 μg of linear DNA) were added to 150 μl of the following reaction mix: 100 mM Tris–HCl pH 7.5, 10 mM MgCl2, 0.2 mM of each four dNTPs, 1 mM NAD+, 15% (w/v) PEG-8000, T5 exonuclease (2 U/ml), Phusion DNA polymerase (33 U/ml); Taq DNA ligase (1666 U/ml), and DpnI endonuclease (50 U/ml). After mixing, the solution was directly incubated for 1 h at exactly 50  C. (c) Before electroporation, 20 μl of the DNA solution was placed on a Millipore VSWP02500 membrane (25 mm diameter, 0.025 μm porosity) floating on a petri dish filled with 40 ml of ultrapure sterile water. Electroporations are then performed classically with 1 μl of DNA for 50 μl of electrocompetent cells.

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a

b

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Phi29 polymerase

ligase

2S-type restriction enzyme

ligase

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Fig. 4 Construction strategy for a library encoding repeat proteins (a) Degenerate oligonucleotides are hybridized and ligated into circles. (b) Each circle from this pool encodes a different repeated motif. (c) The circles are used as a matrix for rolling circular amplification. (d) DNA-homopolymers are digested with a 2S-type restriction enzyme leading to a pool of oriented monomeric double-stranded DNA fragments. (e) Repeats are heteropolymerized into a digested phage-display vector. (f) The ligation product is transformed into electrocompetent bacteria leading to the final library. The library diversity results from the number of inserted motifs, and the sequence of each motif 3.1.2 Library of Proteins with Repeated Motifs [60, 61]

The method is illustrated in Fig. 4. The DNA sequence encoding one motif is assembled using a series of complementary oligonucleotides with degenerate primers for the variable positions. A cassette of “bridging” oligonucleotides complementary to the two extremities of the sequence allows bridging the two ends into circularized double-stranded assemblies. The circles are used as matrix for rolling circular amplification by Phi29 polymerase by using the TempliPhi amplification Kit (GE Healthcare). Large amounts of polymers are obtained, from which monomeric units can be obtained using a 2S-type restriction enzyme. These oriented fragments can be polymerized into the phagemid digested with 2S sites leading to compatible cohesive ends in a Golden Gate-like approach. The resulting population of vectors results in a distribution of sequences with a variable number of repeats.

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The first-generation library is obtained by transforming bacteria with the in vitro prepared DNA. In order to reach the highest possible diversity, multiple electroporations are usually performed, depending on the amount of available DNA. Up to 100 electroporations can be performed in 1 day. After transformation, in vivo fitness biases could favor some clones against others. To reduce these biases and contactdependent inhibition of growth, it is advised to avoid liquid culture for the first-round library production. Hence, the library should be grown on large square petri dishes (400 or 900 cm2) containing agar medium. Not more than 108 individual transformants are grown per petri dish. Typically, 20 μg of vector is used for the transformation of 10–20 electrocompetent cell samples. A detailed protocol for preparing the first-generation library is as follows: 1. After each electroporation, add 900 μl of LB for 100 μl cells. Incubate for 1 h at 37  C. 2. Pool and mix all bacterial suspensions. Take an aliquot of 10 and add 990 μl of LB medium. On small petri dishes, plate 100 μl of serial 10 dilutions of the cells for measuring the library diversity. 3. Spread the electroporation mix on large petri dishes (1 ml/ dish) containing LB-agar and the appropriate antibiotics. Incubate overnight at 37  C or 72 h at 23  C. 4. Recover the bacteria by pouring 30 ml TBS on the agar and resuspending the bacteria. Repeat this once with 10 ml TBS. Pool and mix all the suspensions. 5. Spin down the bacteria at 8000  g for 10 min. Bacteria should be resuspended in LB containing 15% glycerol and stored in aliquots at
80  C.

3.3 Production of a Library of Phage Particles

1. Inoculate in a culture flask a sufficient volume of 2YT supplemented with appropriate antibiotics and glucose with the library of bacteria (starting OD600 nm ¼ 0.1). The volume of the culture depends on the library size; typically, in order to amplify the whole library, the number of living bacteria has to reach a minimum of ten times the diversity. Hence, for a 109 variants library, a volume of 200 ml is usually appropriate. Checking the inoculum’s titer is however advised when starting a selection campaign. 2. Incubate at 37  C upon 200 rpm. 3. When the OD reaches 0.6–0.8, infect the cell with the helper phage in a bacteria:phage ratio of 1:20. The helper phage volume is calculated using the titer of the helper phage in pfu. For the bacteria, an OD600 nm of 1 corresponds to a

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concentration of 5  108 cells/ml. Mix well and incubate the culture at 37  C without agitation for 30 min followed by 30 min under slow agitation (100 rpm) for the infection to occur. 4. Centrifuge the culture for 10 min at 4000  g and resuspend the pellet in the same volume (ex: 200 ml) of 2YT supplemented with antibiotics for selecting both the phagemid and the phage helper, so that only infected bacteria will grow (see Note 2). 5. Incubate overnight at 30  C upon 200 rpm agitation (see Note 3). The phage particles encapsulating the phagemid vectors will be produced in the culture medium. Some particles will expose the protein encoded by the phagemid at their surface. 6. Recover the overnight culture and centrifuge at 12,000  g for 1 h at 4  C. The phage particles are recovered in the supernatant. 7. (Optional) If an amber codon is present in the phagemid between the encoded variants and the coat gene, the suppressive bacterial strain used for the production is not fully efficient and a nonnegligible fraction of proteins will be released in the culture mixed with the phage particles. These potentially functional proteins are not exposed on the phage particles but could infer with the selection process upon binding on the targets. In order to remove these proteins, the purification of the phage particles is necessary. Phage dialysis can be appropriate (see Subheading 3.4.3). 8. The crude or purified phages can be stored a few weeks at
80  C or at 4  C, depending on the stability or the proteins encoded in the library. When using non-phagemid phage libraries, the helper phage infection is not necessary. See Note 4 for more details. 3.4 Purification of Phage Particles

3.4.1 PEG Precipitation

Filamentous phage particles are typically purified by successive polyethylene glycol (PEG) precipitations. Co-precipitation of bacterial products will result in poor purity. The amount of impurities varies with the nature of the displayed protein and with the culture conditions. When high purity is required, a CsCl equilibrium gradient should be performed after PEG precipitations. This will also remove the PEG that can interfere with the binding of phages to some targets. See Notes 5 and 6 regarding protein and DNA stabilities of phages. 1. Spin down a bacterial culture of 250 ml at 12,000  g for 10 min. 2. Carefully transfer 200 ml of the supernatant to a tube containing 50 ml of a solution containing 20% PEG (w/v) and 2.5 M NaCl.

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3. Mix thoroughly and incubate for 1 h on ice. 4. Centrifuge at 12,000  g for 10 min. 5. Carefully discard the supernatant. Centrifuge again at 12,000  g for 1 min and remove the residual liquid. 6. Dissolve the pellet in 20 ml of TBS buffer. Filter on a 1 μm Puradisc 25 GD prefilter unit (Whatman) and then on a 0.45 μm Millex-HV unit (Millipore). 7. Add 5 ml of 20% PEG-2.5 M NaCl, mix well, and incubate for 30 min on ice. 8. Repeat steps 4 and 5. 9. Dissolve the pellet in 1 ml TBS. Add 0.02% NaN3 for longterm storage. 3.4.2 CsCl Equilibrium Gradient

1. Dissolve 2.5 g of CsCl in 3 ml TE. 2. Add the phage solution (1 ml) and adjust the volume to 5 ml with TE. 3. Centrifuge at 200,000  g for 17 h and at 15  C. After centrifugation, the phages appear as a translucent band. PEG appears as a white flocculate below the phage band. 4. Collect the phages by piercing the tube with a needle just below the band and carefully pumping with a syringe. Dialyze twice against 1 L TBS at 4  C for at least 6 h. Add 0.02% NaN3 for long-term storage.

3.4.3 Dialysis

A cellulose ester membrane (Spectrum™ Labs) with large cut-off (300 kDa) will allow the proteins to be exchanged in the dialysis buffer, while the phage particles will be retained in the dialysis internal volume. 1. Place 10–15 ml of phage solution in a dialysis membrane using soft dialysis clips (SnakeSkin™ Dialysis Clips form Thermo Scientific™). 2. Dialyze twice in 1 L of appropriate buffer at 4  C (at least for 4 h and overnight). The buffer will be chosen to be the most appropriate for the target. 3. Recover the phage solution, which is ready to be further incubated with the immobilized target in the panning step.

3.5 Measuring Phage Titer

1. Prepare serial 10 dilutions of the phage solution (see Note 7). 2. Mix 10 μl of these dilutions with 990 μl of a TG1 culture in exponential phase. 3. Incubate at 37  C without agitation for 30 min and under agitation for 30 min.

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4. Spread 100 μl on petri dishes containing the appropriate antibiotic and incubate overnight at 37  C. 5. Count the colonies and calculate the phage titer as colony forming units (cfu). When working with non-phagemid phage library, as high level of display could impair phage infection, we recommend treating the phages with 10
7 M of freshly prepared trypsin (stock at 10
5 M in 20 mM acetate buffer, pH 3.0) for 30 min before measuring the titer. This is not an issue for phagemid libraries. 3.6 Measuring Phage Concentration

Phage concentration is simply obtained by measuring the absorbance at 265 nm and using an appropriate extinction coefficient, which is proportional to the genome size. For a 10-kb phage, the extinction coefficient is 8.4 107 M
1 cm
1. Note that phage concentration determined by absorbance is generally between 20 and 50 times higher than phage titer.

3.7 Evaluating the Level of Display

The level of display is the average number of proteins displayed per phage particle and can be evaluated by western blot. The protocol is a classical SDS-PAGE (10%) followed by transfer on a western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be given to the sample preparation as phages are very stable and difficult to denature. The protocol is similar as a typical SDS-PAGE sample preparation, but β-mercaptoethanol should be replaced by fresh dithiotreitol (DTT, 5 mM final) and the samples should be boiled in a water bath for at least 15 min. Moreover, as the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded on the gel, typically around 1012 phages per lane. The level of protein display is roughly evaluated by comparing the relative intensities of the bands corresponding to the proteinpIII fusion protein and the pIII protein alone, and by considering between three and five copies of pIII per phage particle.

3.8 Rounds of Selection by Affinity Capture

Specific capture of phages on an immobilized target ligand is the most common selection procedure used in phage display. The immobilization step should preserve the structural integrity of the ligand and the accessibility of the interaction site, if defined. The two main immobilization approaches are the unspecific adsorption on plastic wells or tubes, essentially for proteic ligands, and the specific capture of a biotinylated ligand on a streptavidin/avidincoated support like plastic wells, membranes, or magnetic beads. When using biotinylated ligands, the interaction with the phages can be performed in solution, before the immobilization. This provides a better control on the selection pressure by adapting the ligand concentration and may prevent a possible accessibility problem of the immobilized ligand. Moreover, when a protein is

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selected based on its capture on a streptavidin bound ligand, it is not rare that the protein recognizes the streptavidin/ligand complex but not the free ligand. This is especially a problem when working with small molecule ligands. Mixing the phages and the ligand in solution will also more or less prevent this bias. However, the excess of unbound biotinylated ligand should then be removed prior to immobilization, especially when high concentrations are used. Usually, this can be done by PEG precipitation or using a small desalting column. Another way to avoid the selection of phages that are binding to the support is to alternate selection rounds on different supports, for example, streptavidin- and avidin-coated materials, or plastic wells and magnetic beads. The phages (typically around 1012) are then incubated with the support for a defined time (typically between 2 and 24 h). Adding soft detergents (Tween 20 or Triton X-100), albumin, or skimmed milk will reduce nonspecific binding. After the capture, the support is washed, usually between five and ten times with a buffer solution (such as PBS or the most appropriate buffer for the immobilized target) that may contain detergents (e.g., 0.1% tween 20). The bound phages are then eluted by changing the pH, adding a soluble competitor, or cleaving the linker between the displayed protein and the coat protein. If an acidic pH is used, it should be neutralized immediately after elution. If a soluble competitor is used, several hours of incubation is recommended because the dissociation of the phage from the support is slow due to its low diffusion in solution. In some cases, phages are bound so strongly that classical elution is inoperative. Proteolytic cleavage of the displayed protein is then required. Trypsin and factor Xa have been used, but we recommend trypsin since the phage is resistant to this rather unspecific protease. Trypsin will remove the displayed protein only if a cleavable linker is used or if the protein itself is rapidly degraded by trypsin. The elution is then performed with 10
7 M of freshly prepared trypsin (stock at 10
5 M in 20 mM acetate buffer, pH 3.0) for 1 h. Phage elution is followed by bacterial transfection. When working with possible polyvalent phages, removing the displayed protein by proteolysis is recommended prior to infection because polyvalent display on pIII may impair phage infectivity. Phage infection titers should also be measured before (in) and after (out) capture and elution, the ratio out/in being an efficiency indicator of the process (see Note 8). Amplified phages can then be injected into a new round of selection. The increase of the out/in ratio from one round to another is an indication of a successful selection. To progressively increase the selection pressure from one round to another, reducing the time of incubation between the phages and the ligand (immobilized or not) and increasing the number of washings after the capture can be considered (see Note 9). Be aware that binders with less than micromolar affinity cannot be selected by phage display.

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3.9 Screening Individual Clones Following the Selection Step 3.9.1 Production of Monoclonal Phagemid Particles in 96-Well Small-Scale

1. Individual colonies obtained after the selection process are randomly picked in a 96-well culture plate containing 150 μl/ well of 2YT supplemented with appropriate antibiotics and Glucose (and tetracycline for XL1 Blue cells). The plate is agitated overnight at 37  C (600 rpm in an Eppendorf Thermomixer). This culture is the “master plate” and will be stored at
80  C after the addition of 15% glycerol. 2. A new culture plate is inoculated from the matrix plate: for each well, 20 μl of matrix-culture in 140 μl of 2YT supplemented with appropriate antibiotics and glucose. The plate is incubated for 4–6 h at 37  C, 600 rpm. 3. For each well, the culture is infected by 10 μl of helper phage (around 1010 phage particles). The plate is incubated at 37  C for 30 min at 0 rpm followed by 30 min at 300 rpm. 4. 100 μl of this culture is transferred in a 96-well format into a deep-well plate containing 1.5 ml of 2YT/well supplemented with antibiotics for phagemid and helper phage selection. 5. The deep-well plate is incubated overnight at 30  C to let the monoclonal production of the phage particles. 6. The plate is centrifuged at 3000  g for 2 h at 4  C and the supernatant containing the phage particles is used for the Phage-ELISA screening step.

3.9.2 Clonal Phage-ELISA

A monoclonal Phage-ELISA is performed to screen the monoclonal phage particles’ binding properties on the immobilized target. Two immunosorbent microplates (Nunc maxisorp) are used to screen one plate of monoclonal phages. For each line, the phages are transferred to a line containing the target and to a line without the target as a specificity control. The coated and noncoated wells should be in the same ELISA plate for a good comparison of the signals. 1. The target is immobilized on the plate in the same conditions as for the panning. And the plates are blocked with BSA 4% in the same buffer (e.g., TBST) as the one used in the selection. 2. 100 μl of phage solution produced in the deep-well plate are transferred in the ELISA plate in the coated and noncoated wells. The phages are incubated for 1–2 h like in the selection process. The plates are washed four times with 300 μl of washing buffer (e.g., TBST). 3. For each well, 100 μl of horseradish peroxide conjugated antiM13 monoclonal antibody (GE Healthcare) is added and incubated for 1 h. The plates are washed four times with 300 μl of washing buffer.

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4. Bound phages are revealed by the addition of BM Blue POD substrate (Roche Diagnostic) leading to a blue color in the positive wells; the reaction is stopped by the addition of 100 μl HCl 1 M, which leads to a yellow color. Absorbance at 405 nm is measured using a plate reader. 3.10 Competitive Phage-ELISA for Evaluating Affinity Constant

4

If phage particles are not polyvalent, dissociation constants can be determined by competitive ELISA following the procedure described by Friguet et al. [83]. Microplates are coated with the ligand and the range of phage concentrations providing a linear signal, directly proportional to the phage concentration, is determined. A concentration that gives a good signal but for which less than 2.5% of the phages are trapped by the coated ligand is chosen. To measure the dissociation constant, phages at this concentration are incubated with various concentrations of ligand in solution for 1 h at room temperature under stirring. After the equilibrium is reached, the proportion of free phages in each sample is determined on ligand-coated microplates. Since the equilibrium in solution is not significantly modified by immobilization, the ELISA signal is proportional to the free phage concentration. If the phage concentration is much lower than the range of ligand concentrations, a sigmoidal titration curve is obtained. The ligand concentration giving 50% of the signal is equal to the dissociation constant.

Notes 1. If possible, purify any DNA fragments by acrylamide gel electrophoresis. For classical ligation, use a ratio vector/fragment of 1/3. Purify the ligation mix as thorough as possible and concentrate the DNA to approximately 1 μg/μl. For transformation, use large amounts of well-purified restricted vector. We recommend preparing the vector with a Qiagen maxipreparation kit and purifying it furthermore with CsCl gradient. Make a transformation test before creating the library and compare with a standard like pUC18. A reasonable objective is between 106 and 107 transformants per electroporation. Typical libraries are in the order of 107–109 independent clones, while libraries over 1010 independent clones have been described [84]. Special attention must be paid to double transformation artifacts [85, 86]. Combinatorial approaches have also been used to increase library size up to 1012 clones [87– 89]. 2. We recommend using the Phaberge helper phage that carries a kanamycin resistance marker so that the antibiotics can be used for removing noninfected bacteria.

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3. Infected bacteria can produce phages at various temperatures, typically between 20 and 37  C. The rate of phage production will increase with the temperature and with the bacterial density. However, the level of surface display will usually decrease with increasing the growing temperature. This depends on the stability of the displayed protein, since increasing the temperature often results in increasing the sensitivity of the fusion protein to in vivo proteolysis. The time of the culture is also an important parameter, as it must be long enough for the production of sufficient amount of phages. On the other hand, the level of display, that is, the number of properly folded proteins displayed per phage particle, generally decreases with the time of culture. 4. Since phage vectors used in phage display contain an antibiotic resistance marker (tetracycline if working with fd-DOG1 phages), phage particles are produced simply by growing infected bacteria in the presence of the antibiotic. Time, temperature, and inoculum quantity can affect the quality and the quantity of the phages (see Note 3). Every time phages must be produced for selection, it should be prepared from phageinfected bacteria (infected bacteria must be stored in glycerol 40% at
80  C). Typically, phages are produced directly after inoculation by growing infected bacteria for either 20 h at 37  C or 72 h at 23  C with orbital shaking at 180 rpm. Alternatively, an overnight culture at 37  C in 25 ml of LB medium can be centrifuged and resuspended in 250 ml of fresh medium and then incubate for 4 h at 30 or 37  C with orbital shaking at 180 rpm. This will generate less phages but with more reproducibility and generally higher level of display. 5. The displayed protein can be unstable over time. This could be due to the presence of proteases or to inherent low protein stability. Cocktail of protease inhibitors could be added (Complete tabs, Roche). Always use freshly prepared phage solutions when performing selection from libraries. 6. Sometimes, phages are not genetically stable. This could be due to a low toxicity of the fusion protein or to recombination with homologous E. coli genes. Use a recA strain such as JM109 to reduce recombination. For the toxicity problem, use a phagemid vector such as pHDi.Ex [71], which allows control of the fusion protein expression. With this vector, strong repression will be obtained by adding 1% glucose (catabolic repression). The use of the phage shock pPsp promoter can also be envisioned [72]. 7. Phages are sticky toward themselves and toward solid supports. When they are concentrated, they can form soluble aggregates that will dissociate relatively slowly. Therefore, it is recommended to vortex thoroughly the phage solutions before

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transfection. As they will stick to micropipette tips, it is recommended to change tips when performing serial dilutions. Finally, when phages are highly diluted, a time-dependent loss of transfection could result from their binding to vessel walls. It is therefore recommended to use silanized microtubes or to add 1% BSA to the solution or to avoid keeping highly diluted solutions for long times. 8. If the ratio “out/in” is not increasing with the selection rounds, it could mean that no clones are being selected. In case of low-affinity capture, the level of specifically recovered phages might always be below the background level. It is therefore worth analyzing the selected phages, as effective enrichment might have occurred. 9. If a selected binder has insufficient affinities for the desired application, they can be improved by affinity maturation. Mutations can be introduced in the selected clone(s) using several approaches, random or localized: localized rational saturation mutagenesis [90], systematic mutagenesis [91], random mutations (error-prone PCR, mutator strains) [92], or loop or DNA shuffling [93, 94]. Random approaches are generally better suited to improve affinity. Several phage display selection rounds are then performed with increased stringency conditions (koff selection, addition of a soluble competitor, etc.). References 1. Smith GP (1985) Filamentous fusion phage – novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317 2. Leemhuis H, Stein V, Griffiths AD, Hollfelder F (2005) New genotype-phenotype linkages for directed evolution of functional proteins. Curr Opin Struct Biol 15:472–478 3. Sioud M (2019) Phage display libraries: from binders to targeted drug delivery and human therapeutics. Mol Biotechnol 61:286–303 4. Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8:1177–1194 5. Frei JC, Lai JR (2016) Protein and antibody engineering by phage display. Methods Enzymol 580:45–87 6. Hamzeh-Mivehroud M, Alizadeh AA, Morris MB, Church WB, Dastmalchi S (2013) Phage display as a technology delivering on the promise of peptide drug discovery. Drug Discov Today 18:1144–1157 7. Rakonjac J, Bennett NJ, Spagnuolo J, Gagic D, Russel M (2011) Filamentous bacteriophage:

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Chapter 9 Whole-Bacterium Ribosome Display Selection for Isolation of Antibacterial Affitins Ghislaine Be´har, Stanimir Kambarev, Jennifer Jazat, Barbara Mouratou, and Fre´de´ric Pecorari Abstract Classically, in order to perform ribosome display selections, a randomized gene library encoding potential affinity proteins is incubated with a pure target of interest that is either immobilized on a solid surface such as microtiter plates and paramagnetic/agarose beads or used in solution in a way that allows the capture of specific binders. The success of these selections mainly depends on the availability and conformation of the target used. In some cases, the most relevant target can be unknown, or it can be impossible to get it in its native state. Here, we describe a method to perform ribosome display selections against targets at the surface of live bacterial cells. The protocol explains the preparation of bacteria as selection target, panning with libraries, elution, and screenings procedures. We exemplify this strategy with the selection of Sac7dbased Affitins against bacteria as target. Key words Ribosome display, Bacteria, Affitin, Sac7d, Sul7d

1

Introduction Many display methods have been described for the selection of affinity proteins from combinatorial libraries, such as phage display [1], bacterial surface display [2], yeast surface display [3], mRNA display [4], and ribosome display [5, 6]. These techniques have been proven to be powerful tools for identification of candidates able to detect, capture, or inhibit targets, which can be useful for basic research, diagnosis, and therapy. The success of such approaches relies on the use of a target that must be in a state as close as possible to its natural conformation. When the target of interest is a cell-surface protein, it is often mandatory to use a recombinant version of the protein. This can be justified, for instance, to produce a soluble fragment of the protein, or to get

Ghislaine Be´har and Stanimir Kambarev contributed equally with all other contributors. Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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enough material to perform selections and screening steps, and/or to modify it to add a tag for its immobilization on solid surface and to catch it during selections. However, this can be problematic because these methods will select for what is presented. When the recombinant protein is of poor quality with a nonnative conformation, there is a high risk for isolation of binders unable to bind the target in its native state. Additionally, the use of a pure recombinant protein as target can also lead to candidates that bind only epitopes that are inaccessible in the natural environment of the protein, making them useless for subsequent applications. Furthermore, in some cases it can be impossible to have the protein target in a format usable for selections or the target itself is unknown because the corresponding biological system is not sufficiently characterized. Selection strategies based on the use of cells instead of recombinant proteins have been developed to overcome these difficulties. For instance, it has been shown that it is possible to perform selections to isolate peptides and single-chain antibody fragments by phage display [7–9] or yeast surface display [10–12] using mammalian cells as targets and, thereby, surface-exposed proteins in their native physiological context. However, a major drawback of these methods is the need for tedious and inefficient transduction/ transformation in order to enrich either the initial input libraries or the subsequent selection round outputs. Consequently, the sequence diversity at these steps could be significantly reduced or biased. An alternative selection approach which is able to overcome this limitation is ribosome display [5]. In comparison to the mentioned in vivo methods, ribosome display is being performed entirely in vitro, thus avoiding the need for transduction/transformation of living entities. This allows access to sequence diversity of at least 1012 variants [5, 6]. During selections by ribosome display, the link between the phenotype (polypeptide) and the genotype (mRNA) is ensured by the ribosome. In this protocol, we describe a ribosome display selection strategy to target proteins on the surface of living bacteria. As an example, we will explain each step of the protocol with a selection of Affitins, a class of artificial affinity proteins that our group has developed [13–25], with affinity and specificity for bacteria [26, 27]. This protocol can also be performed with other kinds of libraries such as peptides or antibodies.

2 2.1

Materials Strains

1. Bacterial species of interest for selection. 2. Escherichia coli DH5α and DH5 α F’IQ cells (Invitrogen or another supplier).

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2.2

Primers for

2.2.1 Generation of Randomized sac7d Gene Library

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SClib1: GGAGATATATCCATGAGAGGATCGCATCACCATCA CCATCACGGATCCGTCAAGGTGAAATTC. SClib2: GGATCCGTCAAGGTGAAATTCNNSNNSNNSGG CGAAGAAAAAGAAGTGGACACTAGTAAGATC. SClib3: CTTGCCGTTGTCGTCGTASNNAAASNNCACSN NTTTGCCSNNACGSNNAACSNNSNNGATCTTACTAGTGT CCACTTC. SClib4: TAATAACTCTTTCGGGGCATCTTTCTCSNNCA CSNNGCCSNNGCCSNNCTTGCCGTTGTCGTCGTA. SClib5: CCATATAAAGCTTTTTCTCGCGTTCCGCACGC GCTAACATATCTAATAACTCTTTCGGGGCATC. T7C: ATACGAAATTAATACGACTCACTATAGGGAGACC ACAACGGTTTCCCTC. SDA_MRGS: AGACCACAACGGTTTCCCTCTAGAAATAA TTTTGTTTAACTTTAAGAAGGAGATATATCCATGAGAGGA TCG.

2.2.2 Amplification of tolA and Construction of pFP1001_tolA

tolA_coli_F: GAGAAAGGATCCCTTTATATGGCCTCGGGGG CCGAGTTCGAATCTGGTGGCCAGAAGCAAGCTGAAGAG GCGGCAGCG. tolA_coli_R: TGCATTAAGCTTTTTTTCAGCAGCTTCAG TTGCCGCTTTCTTTC.

2.2.3 Amplification of tolA Linker

tolAk: CCGCACACCAGTAAGGTGTGCGGTTTCAGTTGCC GCTTTCTTTCT. SClink: GCGGAACGCGAGAAAAAGCTTTATATGGCCTC GGGGGCC.

2.2.4 Assembly of the Full-Length Ribosome Display Construct

T7B: 50 -ATACGAAATTAATACGACTCACTATAGGGAGACCA CAACGG-30 . tolAk: CCGCACACCAGTAAGGTGTGCGGTTTCAGTTG CCGCTTTCTTTCT.

2.2.5 RT and PCR After Selection

SDA_MRGS: AGACCACAACGGTTTCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATATCCATGAGAGGATCG. SCepRev: TCGGCCCCCGAGGCCATATAAAGCTTTTTCTC.

2.2.6 Sequencing

Qe30for: CTTTCGTCTTCACCTCGAG. Qe30rev: GTTCTGAGGTCATTACTGG.

2.3

PCR

1. Nuclease-free water (various suppliers). 2. Vent DNA polymerase with reaction buffer and 100 mM MgSO4 (New England Biolabs). Store at 20  C. 3. Taq DNA polymerase with reaction buffer and 50 mM MgCl2 (various suppliers). Store at 20  C. 4. dNTP solution: 10 mM of each dNTP (Thermo Fisher Scientific).

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5. DMSO. 6. 1 kb Plus DNA ladder (Invitrogen). 7. 6 Orange DNA loading dye or equivalent (Thermo Fisher Scientific). 8. Agarose (various suppliers). 9. GelGreen nucleic acid gel stain (Biotium). 10. 1 TAE: 40 mM Tris base, 20 mM acetic acid, 1 mM EDTA. 11. Wizard SV Gel and PCR Clean-Up System (Promega). 2.4

Ligation

1. T4 DNA ligase (New Englang Biolabs). 2. BamHI and HindIII restriction enzymes (Thermo Fisher Scientific). 3. FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific). 4. pFP1001 or any vector with unique BamHI and HindIII restriction sites.

2.5 In Vitro Transcription

1. TranscriptAid T7 High Yield Transcription kit (Thermo Fisher Scientific). 2. DNase I, RNase-free (Thermo Fisher Scientific). 3. 6 M LiCl. 4. 70% (v/v) Ethanol. 5. 100% (v/v) Ethanol. 6. 3 M Sodium acetate. 7. NucleoSpin RNA XS purification kit (Macherey-Nagel). 8. NucleoSpin RNA/DNA Buffer Set (Macherey-Nagel).

2.6 In Vitro Translation

1. In vitro translation system (various suppliers or lab-made).

2.7

1. TBS: 20 mM Tris–HCl (pH 7.4), 150 mM NaCl.

Selection

2. 200 mM L-methionine (Merck). Aliquot and store at 20  C.

2. Tween-20 (Calbiochem). 3. BSA (Merck). 4. Washing buffer (WB): 50 mM Tris-acetate (pH 7.4), 150 mM NaCl, 50 mM magnesium-acetate. 5. WBT: WB containing 0.1% Tween-20. 6. WBT-BSA: WBT containing 0.5% BSA. 7. Elution buffer: 50 mM Tris-acetate (pH 7.4), 150 mM NaCl, 20 mM EDTA. 8. 25 μg/μL solution of S. cerevisiae RNA (Merck) prepared in nuclease-free water. Aliquot and store at 20  C.

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9. RNeasy MinElute Cleanup Kit (Qiagen). 10. Reverse transcriptase (Thermo Fisher Scientific or another supplier). 11. RiboLock RNase Inhibitor (Thermo Fisher Scientific). 2.8

Bacteria Growth

1. LB (Luria-Bertani) broth (Thermo Fisher Scientific). 2. 2YT broth (Thermo Fisher Scientific). 3. PBS (Merck). 4. Ampicillin (Thermo Fisher Scientific). 5. Kanamycin (Merck). 6. Glucose (Merck). 7. Glycerol (Merck).

2.9

ELISA

1. Anti-RGS-His6-HRP conjugate (Qiagen). 2. SigmaFAST OPD (o-phenylenediamine dihydrochloride) tablets (Merck). 3. TBS-Tween: 1 TBS containing 0.1% Tween-20. 4. Bugbuster 10 Protein Extraction Reagent (Thermo Fisher Scientific). 5. MaxiSorp plates (Nunc). 6. MultiScreen-GV, 0.22 μm filter plates MSGVN2210 (Merck). 7. NucleoVac 96 Vacuum Manifold (Macherey-Nagel).

2.10 Clone Screening and Sequencing

1. Luria-Bertani (LB) agar Petri plates containing bacteriological agar (1.5%), ampicillin (100 μg/mL), and kanamycin (25 μg/ mL). 2. 2YT growth medium containing ampicillin (100 μg/mL), kanamycin (25 μg/mL), and 1% glucose. 3. Lysis buffer: TBS containing 1 BugBuster solution and 5 μg/ mL DNase I. 4. IPTG: 1 M isopropyl β-D-1-thiogalactopyranoside solution in water (Euromedex).

3

Methods This protocol describes in detail the performance of ribosome display selection against living coccoid bacteria using Sac7d-based randomized library [26] (see Fig. 1). However, it should be noted that in our laboratory, we have successfully performed selections using alternative Aho7c libraries [25], against bacilli (unpublished results). Thus, we anticipate that various types of bacterial cells as

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Fig. 1 Principle for the generation of Affitins with binding activity directed against bacteria. Sac7d structure, drawn using PyMOL software (www.pymol.org), is represented as an example of Sul7d proteins, which can be used to generate such affinity proteins. Randomized residues in Sac7d library are represented as yellow sticks

well as input affinity scaffolds (e.g. Sul7d proteins [28] may be used for this purpose by adapting oligonucleotides used for the generation of the library and its amplification). As the gene encoding Sac7d is quite short (200 bp), the randomized input library can be synthesized using a combination of standard and degenerated oligonucleotides in a single PCR step. The obtained PCR product is then assembled with the 50 and 30 functional regions of the ribosome display construct necessary for in vitro transcription/translation—a T7 promoter, a ribosome binding site, a tolA linker, and stem loops at the ends to stabilize the construct (see Fig. 2 and Notes 1 and 2). In order to generate a reliable source of tolA spacer, we designed a plasmid derived from pFP1001 that encodes the part of the tolA gene that is needed for the ribosome display construct. The tolA gene is directly amplified from E. coli DH5α. The final PCR product is then in vitro transcribed/translated and the obtained pool of ternary complexes (genotype-ribosomephenotype) is used for selections against a target of interest. Depending on the desired characteristics of the binders needed, four to seven rounds of selection are necessary.

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Fig. 2 Scheme of the ribosome display construct used for selection possessing all functional regions required for successful cloning using BamHI/HindIII sites, in vitro transcription, translation, and selection of a potential DNA sequence. T7p T7 promoter, RBS ribosome binding site, RGS-His6 Arg-Gly-Ser-His6 are the starting amino acid residues of the coding sequence, and they constitute a tag for the detection of binders with antiRGS-His6-HRP conjugate antibody, sac7d randomized gene of Sac7d, tolA TolA spacer. Standard and degenerated oligonucleotides used to generate the library in this format are indicated by black and red horizontal arrows, respectively. According to Fig. 1, the following amino acid residues were randomized: 7, 8, 9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44, 46 3.1 Preparation of Input Library 3.1.1 Randomization of the sac7d Gene

1. The input Sac7d-based library is obtained via randomization of 14 amino acid residues in the sac7d gene (K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46) known to interact with double-stranded DNA (see Fig. 1 and Note 3). This is achieved by PCR using a combination of four standard (T7C, SDA_MRGS, SClib1, SClib5) and three degenerated primers introducing NNS triplets (SClib2, SClib3, SClib4) (see Notes 4 and 5). In order to generate a randomized input library, prepare a set of 50-μL PCRs in thinwalled PCR tubes containing 2 pmol of each internal primer (0.2 μL of 10 μM primer SDA_MRGS, SClib1, 2, 3, and 4), 10 pmol of each external primer (1 μL of 10 μM primer T7C and SClib5), 2 μL of dNTPs mix (containing 10 mM of each dNTP), 5 μL of 10 Vent buffer, and 1 U of Vent polymerase and then adjust with nuclease-free water to 50 μL. 2. Amplify under the following conditions: initial denaturation at 95  C for 5 min, 5 cycles of 95  C for 30 s, 50  C for 30 s, and 72  C for 30 s, followed by 30 cycles of PCR of 95  C for 30 s, 64  C for 30 s, and 72  C for 30 s, and final extension of 72  C for 5 min. 3. Analyze the obtained amplicon by running it on 1.5% agarose gel containing GelGreen dye and 1 TAE buffer by loading

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5 μL of reaction mixture mixed with 1 μL of DNA loading dye. Migrate at 150 V in 1 TAE for about 25 min. Use an appropriate DNA MW standard such as 1 kb Plus DNA ladder; the expected amplicon size is 330 bp. If a proper amplicon is observed, migrate the remaining PCR volume on 1.5% gel (see Note 6). 4. Extract the amplicon using Wizard SV Gel and PCR Clean-Up System kit according to the manufacturer’s recommendation and elute the purified DNA in 30 μL of water. 5. Analyze the eluate on 1.5% agarose gel for quantification. Usually, a microgram of purified amplicon is being obtained if four 50 μL PCRs are performed (see Note 7). 6. In order to check its quality, the library needs to be cloned into the pFP1001 vector using BamHI and HindIII restriction enzymes (see Note 8). To do this, digest 100 ng of purified library with BamHI and HindIII enzymes for 2 h at 37  C. 7. Mix the digested product (200 bp) with the appropriate volume of 6 loading buffer and gel purify the DNA as described in steps 3 and 4. 8. Digest 500 ng of pFP1001 DNA with BamHI and HindIII for 2 h at 37  C. Dephosphorylate 50 and 30 ends with FastAP phosphatase for 30 min at 37  C and inactivate the enzymes for 15 min at 65  C. This digested vector is ready to be used for ligation without further purification. 9. Ligate the purified DNA product in the pFP1001 vector predigested with BamHI and HindIII. Mix 20 ng of the fragment library, 100 ng of the pFP1001 vector, 1 μL of 10 T4 DNA ligase buffer and nuclease-free water to 10 μL. Add 2.5 U of T4 DNA ligase and incubate at room temperature for 60 min. Inactivate the ligase for 10 min at 70  C. 10. Transform E. coli DH5α cells to obtain individual clones and sequence some dozens of them using the Qe30for and Qe30rev primers to ensure that the library was synthesized as designed, and there is no strong bias in the nucleotide composition (see Note 9). 3.1.2 Generation of the pFP1001_tolA Vector

1. Streak E. coli DH5α culture on an LB/agar Petri dish in order to obtain isolated colonies. Incubate overnight at 37  C. 2. Using a sterile tip, pick a colony and inoculate a PCR mix containing 10 pmol of each primer (1 μL of 10 μM primer tolA_coli_F and tolA_coli_R), 4 mM MgCl2 (4 μL of 50 mM), 1 μL of dNTP mix (containing 10 mM of each dNTP), 5 μL of DMSO, 5 μL of 10 Taq buffer, and 2 U of Taq polymerase and then add nuclease-free water to 50 μL.

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3. Place the tubes on a thermocycler and preheat the reaction to 95  C for 5 min. Perform 30 cycles of PCR of 95  C for 30 s, 50  C for 30 s, 72  C for 30 s, followed by 1 cycle of 72  C for 5 min. 4. Mix 1 μL of 6 loading buffer with 5 μL of the PCR product and run on a 1.5% agarose gel to check that the PCR reaction gives a band corresponding to the expected size of 327 bp as described in Subheading 3.1.1, step 3. 5. Cleanup the remaining PCR product (45 μL) using Wizard SV Gel and PCR Clean-Up System kit. 6. Digest 100 ng of the purified DNA with BamHI and HindIII enzymes for 2 h at 37  C. 7. Gel purify the DNA as described in Subheading 3.1.1, step 4. 8. Ligate the purified DNA product with the pFP1001 DNA predigested with BamHI and HindIII (from Subheading 3.1.1, step 8). The reaction contains 100 ng of the pFP1001 vector, 20 ng of DNA insert, 1 μL of 10 T4 DNA ligase buffer, and nuclease-free water up to 10 μL. Add 2.5 U of T4 DNA ligase and incubate at room temperature for 60 min. 9. Inactivate the ligase for 10 min at 70  C. 10. Transform E. coli DH5α cells to obtain individual clones and sequence some of them using the Qe30for and Qe30rev primers as in Subheading 3.1.1, step 10. 11. From one clone with the correct sequence, prepare a stock of purified pFP1001_tolA vector by carrying out a plasmid miniprep according to the manufacturer’s specifications. 3.1.3 Generation of the tolA Fragment

1. Large quantities of tolA spacer can be obtained quickly via PCR amplification from pFP1001_tolA vector. For this purpose, prepare a series of at least ten PCR reaction mixes containing 25 pmol of each primer (0.25 μL of 100 μM primer SClink and tolAk), 20 ng of pFP1001_tolA vector (0.2 μL of 100 ng/μL), 1 μL of dNTP mix (containing 10 mM of each dNTP), 5 μL of 10 Vent buffer, and 1 U of Vent polymerase and then add nuclease-free water to 50 μL. 2. Place the tubes on a thermocycler and preheat the reaction to 95  C for 5 min. Perform 30 cycles of PCR of 95  C for 30 s, 55  C for 30 s, 72  C for 30 s, followed by 1 cycle of 72  C for 5 min. 3. Pool the tubes and gel purify on a 1.5% agarose gel as described in Subheading 3.1.1, steps 3–11. The PCR reaction should give a band of 333 bp. 4. Determine the concentration of the tolA linker by measuring the optical density at 260 nm and considering that a 50 μg/mL

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double-strand DNA solution has an optical density of 1 (for 1 cm light path). Adjust the concentration to 80 ng/μL. This is the stock that will be used for the construction of the library and during selection. 3.1.4 Assembly of the Ribosome Display Construct

1. Prepare a series of PCR reaction mixes in PCR tubes (up to 10) containing 25 pmol of each primer (0.25 μL of 100 μM primer T7B and tolAk), 40 ng of sac7d library (2.7 μL of 15 ng/μL) from Subheading 3.1.1, step 4, 53 ng of tolA linker (0.66 μL of 80 ng/μL) from Subheading 3.1.3, step 4, 1 μL of dNTP mix (containing 10 mM of each dNTP), 5 μL of 10 Vent buffer, and 1 U of Vent polymerase and then add nuclease-free water to 50 μL. 2. Place the tubes in a thermocycler and use the following PCR program: an initial denaturation step at 95  C for 30 s, followed by 8 cycles of 95  C for 30 s, 45  C for 30 s, 72  C for 50 s, followed by 30 cycles of PCR of 95  C for 30 s, 55  C for 30 s, 72  C for 50 s, and by 1 cycle of PCR of 72  C for 5 min. 3. Pool the tubes and check on a 1.5% agarose gel that there is only one PCR product of 635 bp as described in Subheading 3.1.1, step 3 and cleanup the PCR product using Wizard SV Gel and PCR Clean-Up System kit. If there are additional products, perform gel extraction of the band with the right size. Each μg of the obtained library is equivalent to about 1.5  1012 molecules (see Note 10). The library can be stored for several months to years at 80  C.

3.2 In Vitro Transcription

1. Use the TranscriptAid T7 High Yield Transcription Kit from Thermo Fisher. Prepare the transcription reaction following the provider protocol with reagents warmed at room temperature (except enzyme) using 1.2 μg of the prepared DNA library from Subheading 3.1.4, step 3 (6 μL of 200 ng/μL). Incubate at 37  C for 2 h (see Note 11). The transcription mixture should be turbid after about 10 min. Lack of turbidity indicates failure of the transcription. 2. Treat the transcription reaction (20 μL) with 0.5 μL RNA-free DNase I (Thermo Fisher Scientific, 50 U/μL) for 20 min at 37  C in order to eliminate the DNA input. 3. Add 2 μL of 0.5 M EDTA to inactivate DNase I. Subsequently, purify the RNA using the following standard LiCl precipitation method: 1. Add 180 μL of ice-cold nuclease-free water and 200 μL of 6 M LiCl. 2. Vortex and incubate on ice for at least 30 min.

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3. Centrifuge at 20,000  g for 30 min at 4  C and discard the supernatant. 4. Wash the pellet two times with 500 μL 70% ice-cold ethanol and dry it for 5 min in a vacuum concentrator. 5. Dissolve the pellet with 200 μL of ice-cold nuclease-free water by pipetting up and down. 6. Centrifuge at 20,000  g for 5 min at 4  C. 7. Transfer 180 μL of the supernatant to a new tube, add 20 μL of 3 M sodium acetate and 500 μL 100% ice-cold ethanol, vortex, and incubate at 20  C for minimum 30 min or overnight. 8. Centrifuge at 20,000  g for 30 min at 4  C and discard the supernatant. 9. Wash the pellet two times with 500 μL 70% ice-cold ethanol. 10. Centrifuge at 20,000  g for 5 min at 4  C and discard remaining ethanol with a pipette. 11. Dry the pellet for at least 5 min in a vacuum concentrator. 12. Dissolve the pellet in 52 μL of ice-cold nuclease-free water by pipetting up and down; the concentration should be around 2.5 μg/μL. For long storage keep at 80  C. 13. Determine the concentration of the obtained mRNA by UV absorbance and control the quality on agarose gel using the 2 RNA Loading Dye and RiboRuler High Range RNA Ladder from the TranscriptAid T7 High Yield Transcription Kit. 14. Purify the mRNA using a NucleoSpin RNA XS Kit in combination with NucleoSpin RNA/DNA Buffer Set (MachereyNagel) according to the manufacturer’s specifications and measure the concentration. Dilute the mRNA with nuclease-free water to a final concentration of 2.5 μg/μL. Aliquot and store at 80  C. This step ensures template-free RNA, which is crucial for efficient progression of the selection. 15. Check the removal of template by performing a PCR as in Subheading 3.3, steps 17–19 in a volume of 20 μL using 0.5 ng of purified mRNA and 25 cycles of amplification (no PCR product expected). 3.3 Ribosome Display Selection in Bacterial Suspension

1. Grow bacteria in 5 mL media overnight at 37  C with shaking at 150 rpm. 2. Pellet 1  109 bacteria per selection in a sterile 1.5 mL tube for 30 s at 4500  g at 4  C. 3. Carefully discard the supernatant and wash three times with 300 μL of 1 PBS. Make sure not to lose the pellet and that no clumps of bacteria remain after resuspension (see Note 12).

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4. Wash the bacteria once in 300 μL of WBT (see Note 13) and then resuspend them again in 200 μL WBT. Keep the obtained suspension at 4  C on ice until needed. 5. Prepare the translation reaction: each 55 μL reaction contains 1 μL of 200 mM methionine, 25.2 μL of Premix, 25 μL of S30 extract, and 5 μg of mRNA from Subheading 3.2, step 14 (see Note 14). 6. Translate for 8 min at 37  C (see Note 15) and then add 240 μL of ice-cold WBT to stop the translation reaction. 7. Immediately centrifuge for 5 min at 20,000  g and at 4  C and place on ice until needed. 8. Add 300 μL of the stopped translation reaction to the 200 μL of precooled bacterial suspension from step 4. 9. Incubate for 30 min at 4  C under gentle shaking. 10. Pellet the cells for 30 s at 4500  g and 4  C, discard the supernatant, and resuspend the pellet in 500 μL of ice-cold WBT-BSA. 11. Repeat steps 9 and 10 three times during the first selection round. 12. To elute the selected mRNA after the final wash, resuspend the bacteria in 200 μL of ice-cold Elution Buffer containing 50 μg/mL S. cerevisiae RNA and incubate for 10 min under gentle shaking at 4  C. 13. Centrifuge for 1 min at 4500  g and 4  C to pellet bacteria. 14. Purify the mRNA contained in the supernatant using RNeasy MinElute Cleanup Kit (Qiagen, elute mRNA with 25 μL Elution Buffer) according to the manufacturer’s specifications. 15. Prepare a PCR tube per eluted sample containing 24 μL of eluted purified mRNA (from step 14) and 44 pmol primer SCepRev (0.44 μL of 100 μM). Incubate at 70  C for 5 min and, immediately after denaturation, chill on ice. 16. Prepare the reverse transcription mix; per reaction add 8.7 μL of 5 Reverse Transcription buffer, 4.4 μL of dNTPs mix (containing 10 mM of each dNTP), 2.7 μL of Ribolock (40 U/μL), 2.2 μL of reverse transcriptase (200 U/μL), and nuclease-free water to 20.7 μL. Add the denatured mRNA (from step 15) to the reverse transcription mix and incubate at 42  C for 1 h in a PCR machine. Stop the reaction by heating at 70  C for 10 min. Chill on ice. 17. To amplify the reverse transcription products, prepare a series of four PCR reaction mixes in tubes containing 25 pmol of each primer (0.25 μL of 100 μM primer SCepRev and SDA_MRGS), 5 μL of the reverse transcription template (from step 11), 1 μL of dNTP mix (containing 10 mM of

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each dNTP), 1.5 μL of MgSO4, 2.5 μL of DMSO, 5 μL of 10 Vent buffer, and 1 U of Vent polymerase and then add nuclease-free water to 50 μL (see Notes 16 and 17). 18. Place the tubes on a thermocycler and preheat the reaction to 95  C for 5 min. Perform 5 cycles of PCR of 95  C for 30 s, 55  C for 30 s, 72  C for 30 s, followed by 40 cycles of PCR of 95  C for 30 s, 64  C for 30 s, 72  C for 30 s, and by 1 cycle of 72  C for 5 min. 19. Run a 1.5% agarose gel to be sure that the PCR reaction gives a band corresponding to the expected size of 314 bp (see Note 18) as described in Subheading 3.1.1, step 3. 20. Purify the product with Wizard SV Gel and PCR Clean-Up System kit. 21. Determine the concentration of the purified PCR product by UV absorbance as in Subheading 3.1.3, step 4 and store the product at 20  C or at 80  C for several months. 3.4 Additional Selection Rounds

To proceed with an additional round of selection, the promoter and the spacer regions must be reincorporated into the selection output as in Subheading 3.1.4. 1. For this purpose, prepare a PCR reaction mix in a tube containing 25 pmol of each primer (0.25 μL of 100 μM primer T7B and tolAk), 300 ng of PCR template (from Subheading 3.1.4, step 21), 320 ng of tolA linker (4 μL of 80 ng/μL from Subheading 3.1.3, step 4), 2 μL of dNTP mix (containing 10 mM of each dNTP), 5 μL of DMSO, 10 μL of 10 Vent buffer, and 2 U of Vent polymerase and then add nuclease-free water to 100 μL. 2. Place the tubes on a thermocycler and preheat the reaction to 95  C for 5 min. Perform 8 cycles of PCR of 95  C for 30 s, 45  C for 30 s, 72  C for 50 s, followed by 32 cycles of PCR of 95  C for 30 s, 55  C for 30 s, 72  C for 50 s, and by 1 cycle of 72  C for 5 min. 3. Run a 1.5% agarose gel to check that the PCR reaction gives a band corresponding to the expected size of 635 bp as described in Subheading 3.1.4, step 3. Cleanup the PCR product with Wizard SV Gel and PCR Clean-Up System kit and measure its concentration as in Subheading 3.1.3, step 4. The final concentration should be around 150–200 ng/μL. The PCR product is now ready for in vitro transcription for a further round of selection as described in Subheadings 3.2 and 3.3 or can be stored at 80  C for several months to years. 4. Repeat the round as described in Subheading 3.3 (see Note 19).

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3.5 Follow-Up of the Selection

A decreasing number of PCR cycles needed to obtain the same quantity of RT-PCR product from round to round is a good indication for an enrichment of the selection. Usually, a decrease of 5 cycles for every round of selection is observed (see Note 20). To test whether the selected Affitins bind specifically to bacteria, it is essential to carry out a whole-cell ELISA (see Note 19) either by coating the bacterial species of interest on 96-well microtiter plates (see below) or, alternatively, by using bacterial suspensions and 96-well filter plates (see Note 21) when the bacteria cannot be coated on polystyrene surface. This can be done by the following procedure: 1. Coat 108 bacteria in 100 μL 1 PBS per well of a Maxisorb plate for 2 h at 25  C with shaking (see Note 21), as well as wells for negative control with irrelevant bacteria. 2. For each selection round, translate the output pools in vitro for 1 h at 37  C (see Subheading 3.3). 3. Dilute the translations eight times with the appropriate volume of 1 TBS containing 0.1% Tween (see Note 13) and distribute 100 μL of this mixture into each test well. 4. Incubate for 1 h at RT with agitation; wash with 6  300 μL of 1 TBS-Tween (see Note 13). 5. Distribute 100 μL of anti-RGS-His6-HRP conjugate (1/10,000) in each well; incubate for 1 h at RT with agitation. 6. Wash with 6  300 μL of TBS with Tween 0.1% and distribute 100 μL of OPD substrate 1 mg/mL and read the optical density value at 450 nm (see Note 22).

3.6 Clone Screening and Sequencing

Pools of sequences that have been selected can be analyzed as follows: 1. Subclone the RT-PCR product from a selection round into pFP1001 via BamH1 and HindIII restriction sites according to Subheading 3.1.2, steps 6–10. 2. Transform E. coli DH5αF’IQ cells with the ligation product, and plate on LB/agar/ampicillin/kanamycin Petri plates to obtain individual clones. Incubate overnight at 37  C. 3. Affitins from isolated clones are then produced in a 96-well deep well culture plate as follows. 4. Distribute with a step-pipette 1.4 mL/well of 2YT containing ampicillin/kanamycin/1% glucose. 5. Inoculate each well with a colony from the plate. 6. Seal the deep-well with a gas permeable adhesive and incubate overnight at 37  C with shaking at 750 rpm.

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7. Add glycerol to a final concentration of 16% to each well, homogenize with shaking. 8. This master plate can be kept for long-term storage at 80  C. 9. For the production of Affitins, distribute with a step-pipette 1.2 mL/well of 2YT containing ampicillin/kanamycin/0.1% glucose. 10. Inoculate with a multichannel pipette each well with 200 μL from the master plate. 11. Incubate 3 h at 37  C with shaking at 750 rpm (the medium should be turbid). 12. Induce the production of Affitins with 0.5 mM IPTG and incubate 3 h or overnight at 30  C with shaking at 750 rpm. 13. Centrifuge the deep-well plate for 20 min at 2000  g. 14. Discard supernatants by flicking the deep-well quickly on top of a trash. 15. Add 50 μL per well of lysis buffer and shake the plate for 30 min at 2000  g and RT. 16. Add 250 μL per well of 1 TBS and shake the plate for 30 min at 2000  g at RT. 17. Centrifuge the deep-well plate for 20 min at 2000  g at RT. 18. These crude extracts from clones are then screened by an ELISA test as in Subheading 3.5 by using 100 μL per well (see Note 22). 19. Use Qe30for and Qe30rev primers to sequence positive clones.

4

Notes 1. The tolA spacer allows the displayed proteins to exit from the ribosome translation tunnel allowing their proper folding away from the ribosome with enough degree of freedom to interact with the target. 2. This construction is adapted from the one described by Amstutz et al. [29] with modifications to fit our needs, such as different detection tags and restriction sites. This system uses prokaryotic in vitro translation with an E. coli S30 extract for ribosome display. (Fig. 2). 3. This mutagenesis scheme is focused on amino acids in the DNA-binding area of the Sul7d family. This surface is large enough, about 1000 A˚2, and slightly concave to match shapes of most globular proteins. 4. For the construction of high-quality libraries, it is recommended to use highly purified oligonucleotides (HPLC or gel

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purification) to avoid as much as possible undesirable sequences due to n1 products. 5. NNS (N ¼ A, C, T or G and S ¼ C or G) codons used for the mutagenesis encode all amino acids while minimizing the number of stop codons. Other mutagenesis schemes can be used depending on needs. 6. This DNA product corresponds to the sac7d gene, flanked by the 50 sequence necessary for ribosome display and an additional 30 sequence necessary for subsequent PCR assembly step with the tolA spacer (Fig. 2). 7. The upper limit for the size of the library at this step can be estimated to 1012 variants when manipulating 1 μg of DNA. 8. pFP1001 is an expression vector derived from the Qiagen vector pQE30 in which we have replaced the suppressible stop of the original vector with two nonsuppressible stops. This allows expression in the DH5αF’IQ. However, pQE30, or any other vector with unique restriction sites BamHI and HindIII, can be used instead of pFP1001. 9. Usually the sequencing of some dozens of randomly picked clones confirms that the observed residue frequency is similar to the predicted one. Alternatively, one can use next-generation sequencing to evaluate quality of the library. It is important to evaluate the quality of the library as it will determine the “useful diversity” and thus the chances of successfully selecting for binders. 10. The quality of the PCR product is crucial for the efficiency of the subsequent in vitro transcription. When a single sharp band without any smear appears on agarose electrophoresis, use this product without further gel-extraction as the template for in vitro transcription. 11. Calculate the volume of the transcription reaction required to retain the library complexity. Generally, most of the product is generated within 1–2 h; however, longer incubations can yield more product. Several basic precautions including wearing gloves, using disposable materials and filter tips, and tubes purchased RNase/DNase-free are required when working with RNA. Water can be DEPC-treated. A water purification system producing RNase-free water can also be used. 12. For bacteria prone to agglutination, a mild sonication treatment in a water bath (1–5 min) can help to disperse bacteria before use. 13. When necessary, other detergents which are known to be milder than Tween 20 can also be used to minimize loss of noncovalently surface-exposed proteins.

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14. We use an E. coli S30 extract and Premix prepared according to the procedure of Amstutz et al. [29]. This extract is prepared from MRE600, an E. coli strain that is deficient for RNAse I. Other commercially available translation mixes can be used when only a few translation reactions are performed. 15. For each new batch of E. coli S30 extract and Premix, the duration of the translation should be optimized. Usually, translation times between 5 and 10 min give optimal yields. 16. Perform the RT-PCR reaction at least in quadruplicate in order to maintain variability of selected sequences. It is important to prepare negative control reaction by replacing the mRNA template with water to detect a contamination of reagents; this control will be checked by PCR in the next step (see Note 20). 17. Prepare negative control reaction by replacing the RT template with water to detect contamination of PCR solutions. Test also the RT negative control prepared in the previous step (see Note 16). If a band is observed in the negative reaction, discard all PCR solutions and repeat the selection round. 18. When a diffuse band or other side-products appear on agarose electrophoresis, gel-purify the band of interest and use this product to initiate a second PCR. This will normally yield a high-quality DNA. 19. Some bacteria such as Staphylococcus aureus express antibody binding proteins at their surface (SpA and Sbi for S. aureus). This makes impossible the use of antibodies to probe binding of Affitins to these bacteria. Thus, consider modifying Affitins to display a biotinylated AviTag sequence at their C-terminal allowing detection with a streptavidin horseradish peroxidase conjugate (streptavidin-HRP conjugate), for instance [26]. 20. The number of RT-PCR cycles needs to be adjusted for each selection round. If too many cycles are done, this will normalize the relative proportions of different pool members, reducing the selective enrichment due to binding. A general rule is to reduce by about 5 cycles per round. 21. It is possible that the bacterial species of interest cannot be coated on polystyrene microtiter plates even when derivatized surface is used. In such cases, it is convenient to use filter plates that allow the performance of ELISA on bacterial cells in suspension. If this is the case, set the follow-up ELISA according to Subheading 3.5 but use MultiScreen-GV, 0.22 μm or 0.45 μm filter plates instead of standard flat-bottom polystyrene ones. At the two washing steps (steps 4 and 6), perform the washes by filtering the bacterial suspension rather than

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simply removing the washing buffer by flipping the plate, since this will lead to loss of cells. This is achieved by mounting the filter plate on a NucleoVac 96 Vacuum Manifold and applying vacuum after each wash. 22. The ratio of (OD450 for the target)/(OD450 for the negative control) should be at least 5–10 for a successful result.

Acknowledgments The authors thank all previous members of the laboratory who helped to develop this protocol. Ghislaine Be´har and Stanimir Kambarev contributed equally to this work. References 1. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554 2. Fuchs P, Breitling F, Dubel S, Seehaus T, Little M (1991) Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein. Biotechnology (N Y) 9:1369–1372 3. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557 4. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94:12297–12302 5. Mattheakis LC, Bhatt RR, Dower WJ (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A 91:9022–9026 6. Hanes J, Pluckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A 94:4937–4942 7. Barry MA, Dower WJ, Johnston SA (1996) Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 2:299–305 8. de Kruif J, Terstappen L, Boel E, Logtenberg T (1995) Rapid selection of cell subpopulationspecific human monoclonal antibodies from a synthetic phage antibody library. Proc Natl Acad Sci U S A 92:3938–3942 9. Jones AR, Stutz CC, Zhou Y, Marks JD, Shusta EV (2014) Identifying blood-brain-barrier

selective single-chain antibody fragments. Biotechnol J 9:664–674 10. Wang XX, Shusta EV (2005) The use of scFvdisplaying yeast in mammalian cell surface selections. J Immunol Methods 304:30–42 11. Wang XX, Cho YK, Shusta EV (2007) Mining a yeast library for brain endothelial cell-binding antibodies. Nat Methods 4:143–145 12. Williams RM, Hajiran CJ, Nayeem S, Sooter LJ (2014) Identification of an antibody fragment specific for androgen-dependent prostate cancer cells. BMC Biotechnol 14:81 13. Mouratou B, Schaeffer F, Guilvout I, TelloManigne D, Pugsley AP, Alzari PM, Pecorari F (2007) Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD. Proc Natl Acad Sci U S A 104:17983–17988 14. Krehenbrink M, Chami M, Guilvout I, Alzari PM, Pecorari F, Pugsley AP (2008) Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD. J Mol Biol 383:1058–1068 15. Pecorari F, Alzari PM (2008) OB-fold used as scaffold for engineering new specific binders. Patent Publication Nos. PCT/IB2007/ 004388 16. Buddelmeijer N, Krehenbrink M, Pecorari F, Pugsley AP (2009) Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane. J Bacteriol 191:161–168 17. Cinier M, Petit M, Williams MN, Fabre RM, Pecorari F, Talham DR, Bujoli B, Tellier C (2009) Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-

Whole-Bacterium Ribosome Display Selection based microarrays. Bioconjug Chem 20:2270–2277 18. Miranda FF, Brient-Litzler E, Zidane N, Pecorari F, Bedouelle H (2011) Reagentless fluorescent biosensors from artificial families of antigen binding proteins. Biosens Bioelectron 26:4184–4190 19. Be´har G, Bellinzoni M, Maillasson M, PaillardLaurance L, Alzari PM, He X, Mouratou B, Pecorari F (2013) Tolerance of the archaeal Sac7d scaffold protein to alternative library designs: characterization of antiimmunoglobulin G Affitins. Protein Eng Des Sel 26:267–275 20. Be´har G, Pacheco S, Maillasson M, Mouratou B, Pecorari F (2014) Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability. J Biotechnol 192 (Pt A):123–129 21. Correa A, Pacheco S, Mechaly AE, Obal G, Be´har G, Mouratou B, Oppezzo P, Alzari PM, Pecorari F (2014) Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins). PLoS One 9:e97438 22. Pacheco S, Behar G, Maillasson M, Mouratou B, Pecorari F (2014) Affinity transfer to the archaeal extremophilic Sac7d protein by insertion of a CDR. Protein Eng Des Sel 27:431–438 23. Be´har G, Renodon-Cornie`re A, Mouratou B, Pecorari F (2016) Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins. J Chromatogr 1441:44–51

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24. Fernandes CSM, Dos Santos R, Ottengy S, Viecinski AC, Be´har G, Mouratou B, Pecorari F, Roque AC (2016) Affitins for protein purification by affinity magnetic fishing. J Chromatogr 1457:50–58 25. Kalichuk V, Renodon-Corniere A, Behar G, Carrion F, Obal G, Maillasson M, Mouratou B, Preat V, Pecorari F (2018) A novel, smaller scaffold for Affitins: showcase with binders specific for EpCAM. Biotechnol Bioeng 115:10 26. Behar G, Renodon-Corniere A, Kambarev S, Vukojicic P, Caroff N, Corvec S, Mouratou B, Pecorari F (2019) Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications. Biotechnol Bioeng 116:1844–1855 27. Vukojicic P, Be´har G, Tawara MH, Fernandez-Villamarin M, Pecorari F, FernandezMegia E, Mouratou B (2019) Multivalent affidendrons with high affinity and specificity toward as versatile tools for modulating multicellular behaviors . ACS Applied Materials & Interfaces 11:21391–21398 28. Kalichuk V, Behar G, Renodon-Corniere A, Danovski G, Obal G, Barbet J, Mouratou B, Pecorari F (2016) The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family. Sci Rep 6:37274 29. Amstutz P, Binz HK, Zahnd C, Pluckthun A (2006) Ribosome display: in vitro selection of protein-protein interactions. In: Celis J (ed) Cell biology – a laboratory handbook, vol 1. Elsevier Academic Press, Cambridge, MA, pp 497–509

Chapter 10 CIS Display: DNA-Based Technology as a Platform for Discovery of Therapeutic Biologics Ana Margarida Gonc¸alves Carvalho Dias Abstract CIS display is a cell-free in vitro display technology, based on linear dsDNA templates. This platform allowed for the evolution of peptides, antibody fragments, and protein scaffolds, mainly for therapeutic applications. In this chapter, CIS display application for discovery of therapeutics biologics is revised. In addition, an improved protocol for DNA recovery at the end of each selection round is described. This improved protocol allows to streamline selections using CIS display, thus accelerating the therapeutic discovery of novel biologics relevant for the pharmaceutical industry. Key words CIS display, In vitro evolution, Protein scaffolds, Antibody fragments, Therapeutics, Peptides

1

Introduction In recent years, in vitro evolution methodologies gained interest for discovery of novel protein-based affinity reagents [1]. In vitro methodologies include cell-dependent display (e.g., phage [2], bacterial, and yeast) or cell-free display (e.g., mRNA [3], ribosome [4], and CIS [5, 6]). The aim of these methodologies is to use a natural or a synthetic design of a protein for evolution. This protein can have totally or partially randomized regions, thus generating a library of compounds. These genetically encoded compounds are transcribed and translated in vitro into proteins and remain physically coupled to the protein, thus creating a genotype and phenotype linkage. These proteins are selected against different targets (e.g., relevant therapeutic molecules, enzymes, biomaterials, or others). After a few rounds of selections, proteins binding the target are enriched. The impact of in vitro evolution methodologies on science and industry are so important that it was recognized by the Nobel Prize

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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committee. The 2018 Nobel Prize in Chemistry was awarded to Frances H. Arnold for directed evolution of enzymes and to George P. Smith and Greg Winter for their work with phage display. Affinity reagents can have different applications, such as therapeutic [7, 8], diagnostic [9], and biocatalysis or purification [10]. Therapeutic applications are one of the major markets for new affinity reagents. These can be based on antibodies or proteins scaffolds. Protein scaffolds have numerous advantages over antibodies, as they have a small size, can be produced more costeffectively, and are generally more stable in different conditions of temperature and chemical solutions. Protein scaffolds can be derived from immunoglobulin fragments (e.g., scFv, Fab, VHH) [7] or CH2 domains (commercial name: Abdurin) [11]. Others are of nonimmunoglobulin origin [8] (e.g., peptide aptamers [12], Fn3-derived centyrins [13], affimers [9], DARPins [14], or Pin1 WW domain [15]). 1.1 Planning an In Vitro Selection for Evolution of Proteins

Before starting an in vitro selection campaign different steps need to be taken: (1) identifying the target; (2) defining the selection goal; (3) selecting a protein scaffold; and (4) designing an appropriate selection strategy. Firstly, for therapeutic targets it is necessary to identify some properties, such as protein function, structural information, biophysical properties, role in disease, homologous proteins in the same host (for specificity), and homology to the target’s orthologs in other animals (i.e., to enable meaningful toxicology studies in rodent or nonhuman primates). Secondly, one should define the selection purpose by identifying the function of the protein scaffold, for example, agonist or antagonist effect, delivery of payloads, or even the affinity range. Thirdly, to select a protein scaffold one should have in mind the size and half-life of this scaffold in blood. It is important to design selection and screening cascades focus in the final application, for example, fusing the protein to an antibody (potentially causing steric hindrance) or using it as a payload delivery vehicle (i.e., as targeting moiety in an antibody–drug conjugate [ADC]). Finally, one should define the strategy to select the protein library against the target. Different strategies can be followed, few examples are as follows: (a) Selecting repeatedly against a single target (e.g., soluble proteins [HSA and/or IgG] or cytokines [TNF alpha]). (b) Selecting against two subunits of a single target (e.g., FcRn and/or VEGFR2), when the biological activity requires binding to both subunits or epitopes simultaneously. (c) Selecting against two distinct antigens, or a complex of different proteins:

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– To identify noncompetitive epitopes. For example, to force the identification of binders to new epitope, selections can be conducted against the target while bound to an already known binder to that target. – For cross-reactivity. Human therapeutic compound development nearly always requires that preclinical in vivo testing be performed in one rodent and one nonrodent toxicology species, before first-in human clinical trials can be allowed. Consequently, it is good practice to identify the homology level between a human target with rodent (typically mouse) and nonhuman primate (typically cynomolgus or rhesus monkey). Two strategies can be followed: (1) selecting repeatedly but in parallel against the two or three target orthologs and identify cross-reactive clones by screening for binding against both or all three, respectively; (2) alternating between two versions of the antigen between consecutive selection rounds (R1 on human, R2 on mouse, R3 on human again, etc.). 1.2 In Vitro Selections—CIS Display

CIS display uses a double-stranded linear DNA template for in vitro selections. This template links the library encoding DNA sequence with the following: promoter, repA gene (a plasmid DNA replication initiation protein, as a single contiguous ORF with the DNA library element), and cis and ori sequences the RepA protein docks to (Fig. 1). The fully assembled CIS template dsDNA is then mixed with Escherichia coli (E. coli) lysate, supplemented with amino acids in in vitro transcription/translation buffer. During this incubation and in vitro protein production, nascent library/RepA fusion

Fig. 1 CIS display library assembly. Representation of different components for CIS activity into a sequence: Promoter—sequence for tac promoter; Library—gene sequence codifying protein scaffold; following as a single open reading frame repA—gene sequence codifying for RepA protein, followed by a STOP codon; cis and ori—gene sequence codifying for sequence bound by RepA protein. After initial PCR or restriction digest/ ligation-based assembly of the CIS construct, a library master stock is produced. For details, see [5]. Master stock is amplified using Primer1—Tac6 and Primer2—OriRev, to produce a working stock. This new material is used as template in CIS display selections

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Fig. 2 In vitro selection using CIS display. Includes improved protocol for DNA recovery described in this chapter

protein binds the nearest ori replication sequence available and halts translation. As the nearest cis/ori region is present on the same linear DNA template that encodes the library/RepA fusion protein, binding only occurs in cis and not in trans—allowing for the high fidelity and stable coupling of phenotype (fusion protein) and genotype (dsDNA template encoding the same fusion) required for effective selection [16] (Fig. 2). 1.2.1 Examples of CIS Display for In Vitro Evolution

CIS display selections had led to the identification of several lead molecules currently in preclinical and clinical development. Some examples can be found in the literature, (i.e., peptides [16, 17], VHHs [18], Abdurins [19], Pin1 WW domains [15], and Centyrins [20]).

Peptides

Peptides were the first scaffolds using CIS display for in vitro evolution. Peptides were identified as a possible strategy for targeted delivery of new therapeutic payloads or can act as pharmacologically active molecules in themselves. Peptides have advantages compared with other small molecular weight compounds, such as higher specificity, due to different interactions with the target across a larger surface contact area (e.g., hydrophobic and hydrophilic interactions); and decreased cost of goods due to cost-efficient production by solid-phase chemical synthesis. Compared with large molecular weight molecules such as antibodies, they more readily allow for incorporation of nonnatural amino acids, as well as other chemical moieties. On the downside, peptides have a limited half-

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life in blood plasma due to renal excretion and proteolysis, which historically limited their success and progression as therapeutic molecules [21]. Nevertheless, some modifications can be made to promote their stability with great success, such as cyclization [22]. To overcome proteolysis instability, a dedicated peptide library was constructed and selected using CIS display. The goal was to identify peptide motifs which could impart increased proteolytic stability to an adjacent peptide sequence, without requiring the modification of said peptide sequence. This library included the following elements: FLAG antibody epitope tag (tag for identification); a peptide library sequence of 12 random amino acids; and a thrombin cleavage site all expressed as a single ORF fused to RepA. The authors aimed to identify sequences of 12 amino acids that would protect against thrombin digestion. In Round 1, after in vitro transcription/translation reaction the library was incubated with thrombin at room temperature as low stringency challenge (1 U for 2H). Noncleaved fusion proteins were recovered using an anti-FLAG epitope antibody and rescued using PCR. In Rounds 2–5, to increase stringency, selection inputs were treated with thrombin at a more optimal 37
C temperature, as more stringent selection pressure (0.5 U for 30 min). Final selection round outputs were cloned into M13 phagemid as pIII fusion proteins for convenient screening. Twenty-six individual clone phages were incubated with human thrombin at 0.1 U/ml or human chymotrypsin or trypsin–agarose 0.65 U/ml for 3 or 6 h at 37
C. After these treatments, phages where screened for residual FLAG epitope presence through ELISA using the M2 antibody as capture step and detected using anti-M13 phage antibody. Fourteen clones were shown to retain >70% of their original (undigested reference) binding signal. These hits were sequenced, and 11 clones were chemically synthesized on solid phase, with inclusion of a C-terminal biotin affinity tag. These peptides were challenged and tested again under the same conditions as the phages, and proteolytic stability was confirmed. These peptides were tested in vitro for stability rat plasma and human blood, and those which demonstrated higher stability were further tested in vivo in rats. A maximum peptide half-life of 15 min in vivo could be obtained. This report concluded that sequences rich in polar and/or proline residues protect adjacent sequences against proteolytic degradation by thrombin as well as other proteases [17]. VHH

Antibodies remain the gold standard as biologic therapeutics for treatment of severe autoimmune, inflammatory diseases and in oncology [7]. Since antibodies require costly production methods and facilities, more cost-effective alternatives were sought, such as scFv, Fab, or VHH immunoglobulin fragments. Libraries encoding such antibody variable fragments can be readily selected and optimized in vitro against a wide range of target classes, including some

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not compatible with in vivo antibody selection methods (i.e., highly toxic or nonimmunogenic targets). One antibody fragment that has been studied in great extent is the VHH (15 kDa) commercialized under the Nanobody™ name by Ablynx. Like conventional antibody heavy chain variable regions, these molecules have four conserved framework regions interspersed with three variable CDR loops. Especially CDR3 have been explored to generate libraries, due to its long size [7]. Isogenica generated LlamdA™, a fully synthetic VHH library designed to match natural CDR folding patterns but build from the ground up using COLIBRA™, DNA synthesis technology [23]. In one recent report, VHH binders against the Wnt3-binding domain of LRP5 and LRP6 were generated (low-density lipoprotein receptor-related protein 5/6) [18]. The Wnt signaling pathway is important for regulation of stem cell renewal, as demonstrated by mutations and over activation of Wnt pathway associated to cancer. The aim was to develop ligand blocking VHHs, four different VHH libraries with different CDR3 loop lengths (7–22 residues) were selected against human LRP6 region P3E3P4E4. Increased stringency in the different rounds was achieved by decreasing the amount of antigen. After following five rounds of selection, outputs were cloned into an expression vector, produced and tested in ELISA against the immobilized target. Thirty-three unique sequences were identified, three of which demonstrated potent Wnt3 inhibition in cellular assays. Two of these were successfully cocrystallized with LRP6. This report improves the knowledge regarding usage of VHH as inhibitor of Wnt pathway and suggests possible application in Wnt-hypersensitive tumors [18]. Pin1 WW Domain

The Pin1 WW domain within the Pin1 Prolyl isomerase was selected as a scaffold for a CIS display library. Pin1 WW domain is responsible for recognition of enzyme ligands, and belongs to a family of proteins defined by the presence of the WW domain. This family has a protein length of 42 amino acids, characterized by two highly conserved tryptophan residues. Pin1 has a particular folding with three antiparallel β-sheets and two loops, which has been a model to study protein folding [24, 25]. To generate a library based on the Pin1 WW domain, consensus regions across different species were identified. Nine nonconserved regions in both loops and β-sheets were partially randomized. The library was selected against the extracellular domain of vascular endothelial growth factor receptor 2 (VEGFR2). Round 4 outputs were cloned into a phagemid vector and resulting phages were used in an ELISA against the target. Sanger sequencing yielded 13 uninterrupted open reading frames out of 48 hit clones. Further sequence analysis revealed six unique clones, which were tested for off-target binding using ELISA. Three highly specific clones were produced through solid-phase chemical synthesis and characterized for its folding by

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circular dichroism. WW domains display a very characteristic folding with signal in far-UV, with a maximum of positive ellipticity at ~230 nm. Clone B1 had this characteristic signature at 25
C and showed fully reversible denaturation when thermally denatured to 90
C. Clone B1 had an affinity of 44 nM to VEGFR2, bound VEGFR1 but not VEGFR3. Plasma stability was evaluated through incubation with mouse plasma up to 40 h. While the in vitro halflife was only 7 h for the original linear peptide, cyclized variants extended the half-life up to 18 h. Despite cyclization, both peptides retained the affinity to VEGFR2 of the parental linear peptide. Centyrins

The centyrin scaffold library was developed from a cross-species consensus sequence (“Tencon”) based on fibronectin 3 domain from different organisms. Different libraries were developed from this scaffold by introduction of variable regions into either lateral surfaces or in loop regions. The centyrin scaffold can be expressed with high yields in Escherichia coli, is highly soluble and monomeric, and demonstrates high thermal stability [13, 20]. Currently, centyrins are developed by a number of Johnson & Johnson licensees, including ARO Biotherapeutics. By applying CIS display selections to centyrin libraries of different designs in parallel against the same targets, the optimal centyrin configuration can be developed for each target, that is, c-MET, murine interleukin-17A (IL-17A), and rat tumor necrosis factor alpha (TNF-α). Up to nine rounds of selection were applied to each selection project. Stringency was typically increased from Round 5 on to enrich the pool for high affinity binders, either through selection for fast on-rates or for slow off-rates. This was achieved by lowering the amounts of biotinylated target and reducing the incubation time between target and library. In addition, excess nonbiotinylated target was added to preformed complexes between biotinylated target and library, thus outcompeting the low amounts of biotinylated target for high off-rate library members. Round 5 or round 9 selection outputs were cloned into a cytoplasmic expression vector, and lysates were tested in ELISA for hit identification. Primary hits were then characterized for target binding affinity and inhibition of target activity (i.e., competition for receptor–ligand interactions). This selection strategy allowed for the identification of low pM bioactive centyrins for different targets tested [13]. Centyrins were also reported more recently as delivery systems of conjugated payloads [26], or fused to antibodies [27]. In the first example, the surface of an epidermal growth factor receptor (EGFR) binding Centyrin was mutated in different positions to introduce a free cysteine residue that would allow for conjugation to a payload. These muteins were recombinantly expressed in E. coli, purified and conjugated to different payloads. These conjugates were characterized for their molecular weight, monomeric

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format, EGFR binding affinity, thermal stability, as well as their cytotoxicity properties. This cysteine scanning strategy allowed for the identification of 26 positions where point mutations are possible in centyrin scaffold without affecting its expression, stability, and efficacy [26]. In the second example, different centyrin libraries were selected against the extracellular domain of FcγRIIB. Round 5 outputs were tested for binding to FcγRIIB and the closely related homolog FcγRIIA. Hits were identified by Sanger sequencing, expressed recombinantly and tested for monomeric behavior using analytical size exclusion chromatography. Monomeric proteins were further tested in cell binding assays by flow cytometry. Centyrins showing a positive signal in the cell binding assay were then expressed as fusions with the antibody of interest. Several studies were performed to confirm the best position for conjugation, ensuring antibody production levels and stability, as well as potency, were not affected by this fusion. In conclusion, this study demonstrated that centyrins can be conjugated to antibodies, increasing their activity in cases where biological activity depends on interaction with FcγRII receptors [27]. Overall, these examples demonstrate the applicability of CIS display for in vitro evolution of different scaffolds using a very straightforward process. In Table 1, the different properties of CIS display are compared with other methodologies for in vitro selections.

2

Materials – 1.5 ml tubes or 96 deep well plates (DNase and RNase free). – Neutravidin- and/or streptavidin-coated magnetic beads and magnetic separators. – DNA libraries encoding protein scaffold, prepared for CIS display (see Note 1). – Biotinylated and nonbiotinylated target, optionally also nonrelated negative control protein, biotinylated. – Buffers: PBS—phosphate buffered saline (cat. #P5368, Sigma); Washing Buffer—phosphate buffered saline with 0.1% Tween 20 (Tween 20, cat. #P1379, Sigma); Blocking buffer—phosphate buffered saline, 2% BSA with 0.1 mg/ml herring sperm DNA (bovine serum albumin, cat. #A2153 Sigma and herring sperm DNA, cat. #D1811, Promega) (see Note 2). – Escherichia coli lysate for in vitro transcription/translation (E. coli S30 Extract System for Linear Templates, cat. #L1030, Promega) (see Note 3). – 10 ThermoPol buffer (cat. #B9004S, NEB).

10

∗

Multiple

RT

Multiple days

Note: RT, room temperature; limited by E. coli or yeast transformation efficiency and scale; typically sorted and recovered by regrowing

Yeast [29]

∗∗∗

1–2 days

8∗

4
C

1014

Ribosome [4]

Up to 5

2 day

RT

3∗∗

1011∗

CIS

1 day

Low pH High temperature l Organic solvents Low pH Temperature l Protease digestion l Competition

n.a.

∗∗∗∗

Proteins Peptides Protein Peptides l Biomaterials l Cells

Proteins

Proteins

l

l

l

l

Target

∗∗∗∗

yeast are

Affinity maturation

Early discovery Affinity maturation

Selection stages

dependent on the affinity goal;

∗∗∗

Neutral pH with EDTA

l

l

l

l

Elution conditions

dependent on enrichment;

∗∗

Time per round

Phage [2, 28]

Temperature RT

# Rounds

3–5

Library size

1014

Method

Table 1 Comparison between in vitro evolution methods

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– High Fidelity DNA Polymerase kit. – Primers for rescue of DNA template after each selection round can be found in the literature (Tac6 and OriRev [5]). Primers for final selection round DNA recovery and cloning need to be designed for each scaffold and destination vector sequence.

3

Methods CIS display selection protocol is described in [5, 15, 16]. The major difference presented in this chapter is in the recovery step of the output DNA. In the previous recovery protocol reported after R1 or even each selection round, binders are recovered from the magnetic beads by incubation with warm ThermoPol buffer (65
C, 10 min). Half of the eluted material is used as template for a PCR for amplification of the N-terminal library region using nested primers between promoter and library. This material is used to rebuild the full-length CIS construct by a DigLig reaction with the repA gene sequence, followed by PCR amplification of the fulllength construct using primers from the promoter to the ori. This material is used as a starting material in next selection round. This strategy is quite time-consuming. In the protocol described in this chapter, magnetic beads are recovered after any selection round in ThermoPol buffer at room temperature. This suspension is added directly and in total into the rescue PCR reaction at R1, and half of the volume is used as template in the following rounds. This greatly improves recovery product yields and quality, and it is also far less time-consuming. – For in vitro transcription translation (ITT) reaction: use 3 μg of DNA library and mix with components from E. coli lysate in a total volume of 50 μl; incubate for 1 h at 30
C. – Wash 3 neutravidin beads with washing buffer and incubate the beads with blocking buffer for 1 h at room temperature. – Block ITT reaction (protein–DNA complexes) in 550 μl blocking buffer and incubate 15 min in ice. – For Round 1, add biotinylated target in appropriate concentration to the previous mix and incubate for 1 h at room temperature. – Add neutravidin beads blocked (see Note 4) to capture target– protein–DNA complexes and incubate for 15 min at room temperature. – Wash beads with 600 μl washing buffer to remove unbound or weak binders by washing the target–protein–DNA complexes. Repeat this step five times. Wash one time with PBS. – Recover the beads in 100 μl 1 ThermoPol buffer.

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Table 2 Example of strategy for different selection rounds Round

# Washes

Target

1

Low number

High amount

2

Increase

Decrease by half

3

Increase

Maintain

4

Maintain

Decrease by half

5

Maintain

Maintain

– DNA output is recovered from the magnetic beads by polymerase chain reaction (PCR). PCR master mix is prepared as indicated by the manufacturer with nested primers to amplify full CIS display sequence, and 50–100 μl of beads is used as template in a total volume of 500 μl of PCR mix (see Note 5). PCR reaction can be performed for 20–30 cycles. – Analyze PCR results in 1% agarose gel and purify PCR material accordingly with DNA purity (see Note 5). CIS display in vitro selection strategy can be repeated for any number of cycles, but outputs are typically screened at 4–5 rounds of selection (Table 2). First round will start with a target concentration at least ten times higher than the desired final affinity. Selection conditions should be designed to balance the need for stringent enough conditions to separate binders from nonbinding library members (stronger or weaker binders dependent on the selection goal) but also maintain diversity. After selections, binders are identified through a screening strategy, typically target binding screen such as ELISA, AlphaScreen, or flow cytometry with cell sorting (FACS). If output rates are very high, current generation screening strategies would take too much time and resource to identify binders. In an ideal scenario, we would have a low number of rounds with sufficient stringency to allow for enrichment of binders to “screenable” levels but not so stringent as to lose diversity. This optimal balance would allow for sampling the output and identify examples of different binders with a range of affinities (Fig. 3). 3.1 Screening and Characterization of Selection Outputs

After the last round of selection, DNA encoding the binders are rescued by PCR and cloned into an expression vector. Many vectors contain a purification tag (e.g., 6Histidine-tag) and a tag to allow for efficient detection (i.e., FLAG, c-Myc, V5). These vectors are transformed into E. coli, individual colonies are picked and protein expression is induced. ELISA or AlphaScreen can be used to screen bacteria lysate outputs. In ELISA for example, the lysates will be

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Fig. 3 Selection strategy. Ideal strategy allows for achieving binders against the target through a low number of selection rounds, with moderate stringency, thus achieving a good diversity that can be evaluated and screened by common methods, such as ELISA

tested against the selection target and possibly other antigens. If the selection aims for cross-reactivity, that is, with nonhuman primate orthologs, the binders will be tested for binding to human and nonhuman primate proteins. If the selection is aiming to generate high specificity reagents, screening may include proteins closely related to the target. At least one unrelated negative control target protein is typically included to check for any gross selectivity issues (e.g., human serum albumin, hen egg lysozyme, or whole baculovirus particles). The results are analyzed based on the ELISA signal of each clones and ratio between target(s) over one or more negative controls. This strategy will allow for selecting the strongest, most specific hits to take forward into Sanger sequencing. Unique (nonredundant) sequences are identified and representative clones for each sequence are selected for further characterization studies. Characterization of unique clones can include protein expression in a larger volume and purification; testing for proper solubility, lack of aggregation or unspecific matrix interactions by size exclusion chromatography; and testing for exact molecular weight by mass spectrometry. Furthermore, affinity against the target and thermal stability can be assessed at this stage, for example, using surface plasmon resonance (SPR), biolayer interferometry (BLI) or microscale thermophoresis (MST) and nano-differential scanning fluorimetry (nanoDSF) for stability. After this initial characterization, clones that have a monomeric format, correct molecular weight, and highest affinity can be further studied against the target in a native format using cell-based assays, such as binding or internalization assays. In a cell binding assay, clones are tested against a positive cell line—the target is

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Fig. 4 Workflow after in vitro selection for discovery of therapeutic biologics

expressed on the cell surface, and a negative cell line. Cell assays include an incubation step of the hits on the cell surface, after which there is a washing step and the bound hits are detected with an antitag antibody conjugated with a fluorophore. Stained cells are then analyzed for their fluorescence in a flow cytometer. Unique clones can be tested on cells in one concentration or in a dilution set. This last strategy allows for estimating the affinity on the cell surface and can narrow down the number of clones that can go for other cellbased assays or for in vivo assays (Fig. 4).

4

Notes 1. Protein scaffolds with randomized positions are produced as DNA-based sequences to generate a library. This library can be assembled by different strategies, with randomized oligo-based PCR assembly being the most common and accessible. However, Slonomics or COLIBRA™ DNA synthesis technology [23] allows for superior control over DNA sequence randomization. These advantages allow for avoiding erroneous STOP codon inclusion, better control over amino acid (codon) contribution at any given position, avoiding redundancy in the library (multiple codons encoding the same amino acid at a given position, thus lowering the effective library diversity), reduced protein stability liabilities by selectively removing sequences of two or more amino acids from the library (e.g., isomerization, deamidation, glycosylation, protease sites, oxidation sites, or free cysteine residues). Also, integration of next-generation sequencing (NGS) during library assembly steps and in-process quality assurance is possible. For assembly of the DNA library with CIS display promoter and rep A control elements see [5]. 2. All buffers components should be highest grade, certified DNase and RNase free. Filter all solutions using 0.2 μm filters. Decontaminate all materials and bench with “DNA

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decontamination reagent” (e.g., DNAZap™ PCR DNA Degradation Solutions, Invitrogen, cat. #AM9890) and treat equipment with UV as much as possible, to avoid PCR template contaminations from prior selections carried over. 3. This E. coli lysate contains all the components to transcribe and translate the DNA into a protein. Use the conditions recommended by the manufacturer to mix appropriate ratios of lysate, amino acids, buffer, and DNA template. 4. Magnetic beads should be added in excess to the amount of biotinylated target. For example, add 50 μl Neutravidin beads (ThermoScientific, cat. #78152104010150) when using 200 nM target. Dependent on the selection goal, several selections strategies can be performed in parallel using an automated magnetic bead separation system (i.e., KingFisher instruments, Thermo Scientific). 5. Selection progression is evaluated after PCR recovery using primers Tac6—CCCCATCCCCCTGTTGACAATTAATC and OriRev—TGCATATCTGTCTGTCCACAGG, presented in 50 to 30 . The material is visualized through an agarose gel, whereby the quality of DNA fragments is observed (e.g., band size and purity of DNA in a single band). If just one band with correct size use a PCR cleanup kit to purify the output DNA. However, if there are bands besides the correct size, use a gel extraction method to extract the correct size band and proceed with a kit to purify the output DNA before continue for the following rounds.

Acknowledgments The author would like to acknowledge the critical review of the Dr. Guy Hermans, CSO at Isogenica, LLC (currently CEO at Sapreme Technologies BV, Netherlands). His comments and revision contributed greatly to improve the manuscript. References 1. Jijakli K, Khraiwesh B, Fu W, Luo L, Alzahmi A, Koussa J, Chaiboonchoe A, Kirmizialtin S, Yen L, Salehi-ashtiani K (2016) The in vitro selection world. Methods 106:3–13. https://doi.org/10.1016/j.ymeth. 2016.06.003 2. Barbas CF III, Burton DR, Scott JK, Silverman GJ (2001) Phage display: a laboratory manual, 1st edn. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press 3. Seelig B (2011) mRNA display for the selection and evolution of enzymes from in vitro-

translated protein libraries. Nat Protoc 6:540–552. https://doi.org/10.1038/nprot. 2011.312 4. Plu¨ckthun A (2012) Chapter 1. Ribosome display: a perspective. In: Ribosome display and related technologies: methods and protocols. Springer, New York 5. McGregor D, Odegrip R, Fizgerald K, Hederer R, Eldridge B, Ullman C, Kuhlman P, Coomber D (2010) In vitro peptide expression library 2:1–7

CIS Display: DNA-Based Technology as a Platform for Discovery. . . 6. Goldflam M, Ullman CG (2015) Recent advances toward the discovery of drug-like peptides de novo. Front Chem 3:1–8. https://doi.org/10.3389/fchem.2015.00069 7. Steeland S, Vandenbroucke RE, Libert C (2016) Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today 21:1076–1113. https://doi.org/10. 1016/j.drudis.2016.04.003 8. Sˇkrlec K, Sˇtrukelj B, Berlec A (2015) Non-immunoglobulin scaffolds: a focus on their targets. Trends Biotechnol 33:1–11. https://doi.org/10.1016/j.tibtech.2015.03. 012 9. Ferrigno PK (2016) Non-antibody proteinbased biosensors. Essays Biochem 60:19–25. https://doi.org/10.1042/EBC20150003 10. Dias A, Roque A (2017) The future of protein scaffolds as affinity reagents for purification. Biotechnol Bioeng 114:481–491. https://doi. org/10.1002/bit.26090 11. Gehlsen KR, Gong R, Bramhill D, Wiersma DA, Shaun A, Wang Y, Feng Y, Dimitrov DS, Gehlsen KR, Gong R, Bramhill D, Wiersma DA, Shaun A, Wang Y, Feng Y, Dimitrov DS, Dimitrov DS (2012) Pharmacokinetics of engineered human monometic and dimeric CH2 domains. MAbs 4:466–474. https://doi.org/ 10.4161/mabs.20652 12. Weng C-H, Huang C-J, Lee G-B (2012) Screening of aptamers on microfluidic systems for clinical applications. Sensors (Basel) 12:9514–9529. https://doi.org/10.3390/ s120709514 13. Diem MD, Hyun L, Yi F, Hippensteel R, Kuhar E, Lowenstein C, Swift EJ, O’Neil KT, Jacobs SA, Marx J (2014) Selection of highaffinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions. Protein Eng Des Sel 27:419–429. https://doi.org/10.1093/pro tein/gzu016 14. Plu¨ckthun A (2015) Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol 55:489–511. https://doi. org/10.1146/annurev-pharmtox-010611134654 15. Patel S, Mathonet P, Jaulent AM, Ullman CG (2013) Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display. Protein Eng Des Sel 26:307–315. https://doi.org/10.1093/pro tein/gzt003 16. Odegrip R, Coomber D, Eldridge B, Hederer R, Kuhlman PA, Ullman C,

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Chapter 11 Building Scarless Gene Libraries in the Chromosome of Bacteria Gol Mohammad Dorrazehi, Sebastian Worms, Jason Baby Chirakadavil, Johann Mignolet, Pascal Hols, and Patrice Soumillion Abstract Genetic libraries of gene variants are frequently used in protein engineering or molecular evolution experiments. Expression libraries cloned in plasmids are relatively easy to prepare but have some limitations that could prove problematic, such as the need for antibiotic selection markers or the copy number variability. Thus, there is a need for efficient methods for the construction of chromosomal gene libraries with high diversities. Here, we present two completely different methods for building gene libraries in the chromosome of Escherichia coli and Streptococcus thermophilus, which belong to the gram-negative and gram-positive bacterial clade, respectively. The E. coli method is based on a combination of CRISPR-Cas9 cleavage and λ-Red recombination technologies, allowing for generation of libraries containing up to million independent clones. The S. thermophilus method takes advantage of the high efficiency of natural transformation of specific strains, allowing to reach diversities up to the billion scale. Both methods can be developed so that no genomic scar or selection marker is left after chromosomal insertion of the library, a key feature when designing experiments that should mimic the natural context of a gene. Key words CRISPR-Cas9, Homologous recombination, Escherichia coli, Streptococcus thermophilus, Directed evolution, Genetic diversity

1

Introduction Directed evolution is by now a foundational technology in enzyme and protein research, used to improve a wide array of characteristics such as stability, activity, and selectivity. The award of the Nobel Prize in Chemistry to Frances H. Arnold, Sir Gregory P. Winter, and Georges P. Smith in 2018 has confirmed this status. Directed evolution aims to sidestep the problem of predicting the relationship between the primary sequence of a peptide or protein and its function by harnessing the power of evolution through sequential rounds of diversity generation and artificial selection or screening [1].

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_11, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Directed evolution campaigns are typically performed using libraries of genes carried on plasmids. Working with plasmid libraries has several advantages, notably the ease of DNA cloning, recovery, and sequencing as well as the high expression level of protein, thus facilitating downward purification and characterization. These advantages are even stronger with plasmids featuring high copy number that can reach several hundred molecules per cell. However, it also comes with some potential disadvantages. Maintaining plasmids requires the use of extra antibiotics in the culture medium. In addition, the plasmid carrying the library might be incompatible with other plasmids eventually required for screening or selection. On the other hand, at the protein level, although high expression may be advantageous from an experimental point of view, it is also known that highly expressed proteins evolve slowly due to associated fitness costs such as selection against misfolding [2–4]. Because of the variation in copy number, the tight and tunable control of gene expression level is difficult to achieve with plasmids. It has been shown that having finely regulated expression system is important, which may otherwise skew the results of experiments, for example, when doing genotype–phenotype mapping studies [5]. Additionally, leaky promoter on high-copy plasmids can also be an impediment for the expression and laboratory evolution of cytotoxic proteins, cytotoxicity being obviously a matter of dose. The lower expression of a gene that is cloned in the bacterial genome also make some assays based on protein inhibition easier to carry out [6]. For gene libraries in plasmids, the likelihood of entering different variants in the same cell is higher [7]. Intracellular fitness competition can lead to the purge of some variants and thus introduce bias in the library. Finally, for evolution campaigns where the goal is to evolve an enzyme native to the host organism to simulate a natural evolutionary trajectory, cloning a library in a natural genomic environment and, if possible, without the need of antibiotic selection, is a better choice to mimic the native expression and regulations conditions. There is therefore an increasing interest in methods that can generate chromosomal gene libraries to provide tighter control and repression, easier maintenance of the gene, greater compatibility with plasmid-borne system, and better mimicking of the natural context, even at the cost of a more intricate process of DNA manipulation [5]. Several recently published methods allow for the diversification of genomic loci. The pEvolvR system recently published by Halperin et al. couples an error-prone PolI polymerase mutant with a nickase variant of Cas9 to diversify a specific region of the genome in a continuous manner [8]. However, the target region is quite small (a short window up to 350 bp) and the method does not allow for the insertion of designed libraries. These methods also suffer from having a mutation rate that depends on the distance

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from the cutting site. The multiplexed automated genomic editing method invented by George Church allows for efficient insertion and screening of libraries at different loci, but requires advanced and expensive equipment to overcome the low efficiency of recombination through continuous transformation with a library of synthetic DNA fragments [9]. In this chapter, we will present two methods for the creation of scarless chromosomal libraries in bacteria. The first method relies on the combination of CRISPR-Cas9 with the λ-Red recombineering systems for efficient library cloning into the chromosome of E. coli. The second method relies on the highly efficient natural competence of some specific strains of S. thermophilus enabling easy manipulation of its genome.

2

Materials

2.1 Common Kits and Reagents

GoTaq® Flexi DNA Polymerase from Promega and Q5® HighFidelity DNA Polymerase from New England Biolabs are referred to in this chapter. Monarch PCR & DNA Cleanup Kit (New England Biolabs) is used for gel extraction and purification of PCR products. Midi and Maxi prep kits are used for plasmid purification. All other enzymes are purchased from Promega or New England Biolabs, unless explicitly mentioned otherwise. Gibson Master Mix: 100 mM Tris–HCl pH 7.5, 10 mM MgCl2, 0.2 mM of each four dNTPs, 1 mM NAD+, 15% (w/v) PEG-8000, T5 exonuclease (2 U/mL), Phusion DNA polymerase (33 U/mL), and Taq DNA ligase (1666 U/mL) [10].

2.2 Material for Protocols with E. coli

Lysogeny Broth (LB): 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl. When appropriate, we supplement the growth media with kanamycin (50 mg/L), tetracycline (12.5 mg/ L), or streptomycin (50 mg/L).

2.2.1 Growth Media

SOC Medium: 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO4, 10 mM MgCl2, 20 mM glucose.

2.2.2 Strains and Plasmids

E. coli strain TOP10 is purchased from Invitrogen. pCas and pTargetF plasmids are ordered from Addgene (plasmids #62225 and #62226). The pTarget plasmid with a tetracycline resistance gene is created by amplifying the TetR cassette with primer pair J2 and the pTargetF plasmid with primer pair J1 and ligating both PCR products with the Gibson Master Mix (see Subheading 2.1).

2.2.3 Electroporation

All transformations are performed using a Bio-Rad Gene Pulser Xcell™ electroporator together with 2 mm electroporation cuvettes.

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2.3 Material for Protocols with S. thermophilus 2.3.1 Growth Media

S. thermophilus strain LMD-9 is purchased from ATCC (BAA-491). S. thermophilus cells are grown at 37
C in an anaerobic jar (BBL GasPak Systems; Becton Dickinson, Franklin Lakes, NJ) for solid media, and in Eppendorf (no shaking) for liquid culture in skimmed milk medium (SMM, Campina, Belgium), M17 medium (Difco) or chemically defined medium (CDM) as described in Letort and Julliard [11]. M17 and CDM are supplemented with 1% (w/v) lactose (M17L and CDML, respectively). Supplements were added to the different media at the following concentrations: chloramphenicol (5 mg/L), erythromycin (2.5 mg/L), 5-fluoroorotic acid (5-FOA) (500 mg/L) (Melford, UK). 5-FOA is diluted in DMSO at a concentration of 100 mg/mL. Chemicals are purchased from Sigma-Aldrich, unless otherwise specified [12, 13]. The S. thermophilus genome sequence can be accessed from GenBank with accession no. CP000419 (LMD-9). The gene leuB (3-isopropylmalate dehydrogenase) is annotated as STER_1170 in the genome.

2.3.2 Natural Transformation

All transformations are performed in CDML or SMM, with the latter affording higher transformation rates. Synthetic XIP peptide (NH2-LPYFAGCL-COOH) is ordered from Peptide 2.0 (Chantilly, VA), resuspended at a concentration of 500 μM in 100% DMSO, and stored at 20
C in 20 μL aliquots.

2.4 List of Oligonucleotides

All primers are purchased from Eurogentec (Seraing, Belgium) and are listed in Table 1. The sequences in bold and black are the 30 -end which hybridize to the target, while the red sequences are the 50 overhang regions necessary for fragment assembly. The sequence in italic and blue for primer A fw is the N20-rha complementary to the rhaA locus of the E. coli genome (see Subheading 3.1.2). Annealing temperatures (Ta) were predicted taking only the hybridizing sequences of the primers using the online tool available at http:// tmcalculator.neb.com/.

3

Methods

3.1 Libraries in E. coli Using CRISPR-Cas9 and λ-Red Technologies 3.1.1 Principle

Since its first reported use for genomic editing in 2013, CRISPRCas9 has established itself as the prime technology for lab-scale genome editing [14, 15]. In bacteria, CRISPR-Cas9 can be coupled with the λ-Red recombination system to efficiently introduce foreign DNA fragments in the chromosome [16–18]. When used independently, the λ-Red system usually has relatively low efficiency, necessitating the additional use of a selection marker to select for positive colonies. By using CRISPR-Cas9 to cleave the target chromosomal site, insertion of the foreign DNA through λ-Red-mediated homologous recombination is the only way for the

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Table 1 List of primers PCR/Target to

Primer

amplify

Pair

Sequence (5’→3’)

N20 modification

A

on pTarget

B1 U arm B2

SS-pbpA-6xHis

C

D arm

D1

D2

pTarget-N20

PBP-A gene (ep-

E

F

PCR) pTarget-N20 -U-

G

SS-pbpA-D Modified locus in

H

genome PCR product of

I

primer pair H J1 Resistance change on pTargetF

U-tRNASer Rec

J2

K1

Fw

AGCTAGCTCAGTCCTAGGTATAATACTAGTGCCTGTGGCCTGAATCCCCCGTTTTAGAGCTAGA AATAGCAAGTTAAAA

Rv

ACTAGTATTATACCTAGGACTGAGCTAGCT

Fw

AGGCACTTCCGGCTTGCC

Rv

AAATCTTTTTCATGCGCAAAGCTCCTTTGTC

Fw

CTCGAGTAGGGATAACAGGGAGGCACTTCCGGCTTGCC

Rv

AAATCTTTTTCATGCGCAAAGCTCCTTTGTC

Fw

TTTGCGCATGAAAAAGATTTGGCTGGCG

Rv

CCCATTCAACTCAGTGATGATGATGATGG

Fw

TCATCACTGAGTTGAATGGGCGAAAGCCAATC

Rv

AGCAGCAACTGCGGCACA

Fw

TCATCACTGAGTTGAATGGGCGAAAGCCAATC

Rv

GCAGAAGCTTAGATCTATTAAGCAGCAACTGCGGCACA

Fw

TAATAGATCTAAGCTTCTGCAG

Rv

CCCTGTTATCCCTACTCG

Fw

TTTTAGCGTTTAGCGCATCGGCGGCG

Rv

GGCTTTCGCCCATTCAACTCAGTGATGATGATGATGGTG

Fw

GTTGAATGGGCGAAAGCC

Rv

CGATGCGCTAAACGCTAAAACTAAACCAGCCAGCGCCAG

Fw

TTCAGCGAGTGCTTCAGGA

Rv

AGATCGTGACGCACAATCTC

Fw

CCACCTTTACCCCTAATCC

Rv

ACTGGCGAATGCTGTCGT

Fw

GCCGGGCCACCTCGACCTGAGATGCCGCTCGCC

Rv

GCGCATTGTTAGATTTCATCAGTCGATCATAGCACG

Fw

ATGAAATCTAACAATGCGC

Rv

TCAGGTCGAGGTGGC

Fw

AACTATCATCCGTCATCC

Rv

CCTTATGGGATTTATCTTCCTTACAGAAATAGACAAAGTGTCC

(contiuned)

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D-tRNASer Rec

K2

P32 -cat

K3

PSP-oroP

K4

U-tRNASer Lib

K5

D-tRNASer Lib

leuB-Lib

K6

K7

Fw

CCGGAATAATATTCATTAAATTTTTATAAAATTTGTCTATTTCTGAAAAGG

Rv

GATGGTCGTATTTTGACC

Fw

GGACACTTTGTCTATTTCTGTAAGGAAGATAAATCCCATAAGG

Rv

GCAAATGTACTGTCAAGAATATTCACGTTACTAAAGGGAATG

Fw

TACATTCCCTTTAGTAACGTGAATATTCTTGACAGTACATTTGC

Rv

CCTTTTCAGAAATAGACAAATTTTATAAAAATTTAATGAATATTATTCCGG

Fw

GGCTTTTCAAAACTATCATCCGTCATCC

Rv

GGTTCCTCGGGTAGCGGA

Fw

CATAATTTGTCTATTTCTGAAAAGGAGAGGAGGGG

Rv

GTCTGGGAAATGGTGATATGATGAGTGC

Fw

TCCGCTACCCGAGGAACC

Rv

CCCCTCCTCTCCTTTTCAGAAATAGACAAATTATG

bacteria to repair the double strand break and to survive. Therefore, we avoid the need to transform a selection marker along with our gene of interest [19]. For our libraries, we adopted the genome modification protocol from Jiang et al. [17], which uses a two-plasmid system: pCas, encoding the λ-Red recombination system under the arabinose-inducible ParaBAD promoter as well as Cas9 under its constitutive promoter, and pTarget which encodes the sgRNA targeting the genome (Fig. 1). In addition, pCas includes a kanamycin resistance marker and a sequence encoding a sgRNA that targets the pTarget plasmid under the IPTGinducible Ptrc promoter affording the curing of the plasmid after genome modification. pCas itself has the temperature-sensitive replication origin repA101(Ts) for self-curing. The library building relies on several steps (Fig. 2). First, the chromosomal locus of interest is determined and a Cas9 cutting site is chosen. A sequence encoding the chosen sgRNA is introduced in the pTarget. If the library is to be cloned into pTarget (see below), two homology regions (minimum 500 bp each) corresponding to the sequence on each side of the chosen cutting site and required for the λ-Red recombination are also cloned into pTarget. The activity of the sgRNA can then be tested by a toxicity assay in E. coli (see Subheading 3.1.3). In parallel, a DNA library (Lib) is created by any classical technique (error-prone PCR, incorporation of degenerate oligonucleotides, DNA shuffling. . .) and flanked by two homology regions (U and D for up and down) for chromosomal integration. For creating small size libraries (103–104 transformants), a linear DNA library U-Lib-D can be used directly with pTarget for cotransformation of a pCas-containing strain. For larger libraries (105–106 transformants), the library must be

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Fig. 1 The pCas and pTarget plasmids. (Adapted from [17]) A.

E. coli chromosome

pCas U

N20

D

Transformation with library and sgRNA B.

C. pCas

pCas U

D

U

D

pTarget_N20 U

lib

D

U

lib

D

pTarget_N20_Lib Plasmids curing (1. IPTG; 2. 37 C) D. U

lib

D

Fig. 2 Strategy for the generation of gene libraries in the chromosome of E. coli. (a) A suitable nonessential locus is chosen. Within that locus, a Cas9 site (PAM + N20) is selected with the help of scoring algorithms, and 500 bp homology arms (U and D) as close as possible to the Cas9 site (less than 200 bp is recommended). An example is detailed in Fig. 3. The pCas plasmid is introduced into the strain. (b) For reaching high diversities (105–106 individual transformants), the library is cloned into the pTarget, flanked by the U and D homology arms, together with the sequence encoding the sgRNA. The plasmid library is then introduced into the pCas containing strain, triggering chromosome cleavage. Homologous recombination, facilitated by the λ-Red proteins, is the only way to survive. (c) If high diversity is not required (103–104 individual transformants), a cotransformation with the sgRNA encoding pTarget and the U- and D-flanked library as a linear DNA fragment is a simpler protocol. (d) After transformation, the pTarget_N20 induces (IPTG) the expression of an antipTarget sgRNA encoded into the pCas, and the thermosensitive pCas itself is subsequently cured by growing the library at 37
C

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integrated into the pTarget plasmid, in between the previously added U and D homology arms. Once all parts are assembled, the actual genome modification can take place. Cells containing the pCas plasmid are grown and the expression of the λ-Red recombination enzymes is induced by adding arabinose. The cells are then made competent and cotransformed with the linear DNA library and pTarget plasmid or transformed with the library inserted into pTarget. Cas9 is activated by the sgRNA encoded on pTarget and generates the site-specific double-stranded break. Homologous recombination with U-Lib-D DNA is facilitated by λ-Red system while cells that failed to recombine cannot survive. Following verification of a correct insertion, cells can be grown on IPTG to induce the expression of a pCas-borne sgRNA targeting the pTarget plasmid, resulting in its curing. Finally, growing the cells at 37
C will remove the pCas plasmid due to its temperaturesensitive origin of replication. The loss of antibiotic resistance can be checked after curing. As an example, the following protocols are described for a library of pbpA genes (894 bp, encoding a periplasmic penicillin binding protein) created by error-prone PCR and inserted into the rhamnose operon of E. coli in such a way that the pbpA gene is inserted at the second position of the polycistronic operon, with its ATG at the exact same position as that of the wt rhaA gene (Fig. 3). rhaB

A

U

PrhaBAD

B

rhaB

rhaA

PAM

rhaD

rhaA D

N20

----TGCGCATGACCA--- ---GGCGGGGGATTCAGGCCACAGGC----

U

165 bp

Cas9 cutting site

165 bp

D

Lib

C ----TGCGCATGAAAA---

500 bp

SS

pbpA

His-tag

500 bp

Fig. 3 Example of library design in E. coli. (a) The chosen locus is the rhamnose operon, and the objective is to introduce a library of pbpA genes exactly at the position of the second gene (rhaA). (b) Detailed positions of N20, Cas9 cut site and up and down homology regions (U and D). Depending on the position of the appropriate N20, the distance of U from Cas9 cut site will be imposed by the design (165 bp in this case) and the same distance is chosen for positioning the D arm. (c) Donor DNA cassette of pbpA library generated by error-prone PCR and overlap extension assembly. The gene of interest will replace exactly the beginning part of rhaA gene (350 bp) far enough from promoter (see Note 1). The gene of interest (pbpA) will be inserted with the help of homologous arms (500 bp) through λ-Red recombination, leaving intact the open reading frame (ORF) and stop codon of rhaB and using the start codon of the rhaA gene. The pbpA ORF encodes an N-terminal signal sequence (SS) for periplasmic localization and a C-terminal His-tag for purification. Both sequences were not subjected to the error-prone PCR

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The pbpA gene comprises a sequence encoding the amino-terminal export signal (SS) of the DsbA protein for periplasmic localization and a sequence encoding a carboxy-terminal 6His tag for downstream purification. These external sequences are not targeted by the error-prone PCR. 3.1.2 Selection of sgRNAs and Cloning into pTarget

The Cas9 nuclease requires a guide RNA (sgRNA) that comprises a sequence recognized by Cas9 and a 20-nucleotide sequence (N20) complementary to a region of the genome next to a protospaceradjacent motif (PAM) featuring an NGG sequence. The Cas9 endonuclease cuts three base pairs upstream of the PAM sequence. The choice of an N20 is of paramount importance to the library creation process as the efficiency of the Cas9 nuclease depends on the sgRNA [20]. Several algorithms have been developed to help scientists select a suitable N20 site. Many of them are available online as user-friendly interfaces. We used the Design CRISPR Guides tool on the Benchling platform (http://www.benchling. com), which calculates on- and off-target scores based on algorithms developed by Doench et al. [20] and Hsu et al. [21] respectively. The first 200–300 bp of the rhaA gene of E. coli were selected as input to find the highest-scoring N20. On- and off-scores above 60 and 50 are considered to correspond to appropriate guides for maximizing on-target cleavage and minimizing off-target cleavage, respectively. After selection of an appropriate N20, it is introduced into the pTarget vector by amplifying the whole plasmid with a long primer containing a floating tail with the N20 and a shorter reverse primer complementary to the end of the floating tail. After amplification, 100 ng of purified PCR product (linear plasmid) can be self-ligated by adding 15 μL of Gibson Master Mix, 10 U of DpnI, and ddH2O to 20 μL. After incubation for 1 h at 50
C, 1 μL of the Gibson reaction product can be electroporated in E. coli TOP10 cells [10]. In the example used in this chapter, the primers used to insert the N20-rhaA (GCCTGTGGCCTGAATCCCCC), targeting the rhaA locus, are found in the primer table as primer pair A.

3.1.3 pTarget Validation

To validate the sgRNA, E. coli TOP10 cells containing the pCas plasmid are transformed either with the modified pTarget or with a pTarget expressing a sgRNA that does not target the E. coli chromosome and the ratio of transformants is determined. Since double-strand breaks are lethal to E. coli, a plasmid encoding an adequate sgRNA should not be able to transform E. coli that produce Cas9. We use a pTarget_ALR plasmid (N20: GCTGTCTCTAAACTTAGGGC) as negative control. 1. Start a preculture of E. coli TOP10 containing pCas in 5 mL of LB-kanamycin and incubate it O/N at 30
C.

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2. Inoculate 10 mL of LB-kanamycin with 100 μL of preculture. Grow at 30
C until an OD600 of 0.4–0.6 is reached. 3. Centrifuge down the cells for 7 min at 3000  g. Discard the supernatant and resuspend the cells in 5 mL of cold ddH2O. 4. Repeat step 3. 5. Centrifuge the cells for 7 min at 3000  g. Resuspend the cells in 1 mL of cold ddH2O. Transfer to a microcentrifuge tube. 6. Centrifuge the cells in a benchtop centrifuge at 10,000  g for 10 s. Discard the supernatant and resuspend the cells in 100 μL of cold ddH2O. 7. To 50 μL of the competent cells, add 20 ng of either the pTarget to be tested or pTarget_ALR. Transfer to a 2 mm electroporation cuvette. 8. Electroporate the cells at 2.5 kV. Resuspend the cells in 950 μL of SOC medium. Allow the cells to recover at 30
C for 45 min. 9. Plate 10 and 100 μL of each transformation on LB-Kan-Tet plates and incubate overnight at 30
C. 10. Compare the colony count on both plates. If we see a thousandfold difference in the colony count on both plates, it is considered as adequate. 3.1.4 Donor DNA Cassette Design

In order to favor recombination, relatively long homology regions of 500 bp are used. In order to avoid the presence of a promoter in the donor DNA cassette (see Note 1), we inserted the library in the second gene (rhaA) of the rhamnose operon through recombination. The upstream homology arm (U) is designed by taking the 500 bp immediately upstream of the rhaA start codon. The downstream homology arm (D) is designed by taking 500 bp downstream of the selected N20. The homology regions must be as close as possible from the Cas9 cutting site. Because of library design constraints, in our example (Fig. 3), U and D regions are 165 bp upstream and downstream the N20 sequence. This design allowed for the building of a million clones library. Higher diversities may be reached by reducing these distances whenever possible.

3.1.5 Library Construction

As an example, an error-prone library (Lib) is constructed by running an ep-PCR (protocol in Subheading 3.3) using primer pair F and the pbpA gene as template. Both primers used in ep-PCR should harbor homologous sequences to the U and D arms to allow for the subsequent assembly of the ep-library into the donor DNA cassette.

3.1.6 Linear Cassette Assembly to Generate Small Libraries

The homologous arms, U and D, are produced by Q5-mediated PCR from the genome with primer pair B1 for the U arm and pair D1 for the D arm. Since the signal sequence is not included in the

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library, it must be assembled with the U fragment. An SS-pbpA fragment is prepared by PCR using primer pair C and further fused with U by Gibson assembly (see Note 2). Then, this assembled fragment of U-SS-pbpA is used as the template to amplify U-SS without pbpA by the help of primer B1 forward and G reverse. DNA fragments of U-SS and D harbor overhangs complementary to the extremities of the library. Following purification of the DNA, the library and the two homologous arms should be assembled in a way to minimize the introduction of bias in the donor DNA library. Two different methods described below can be used that give similar results. Primerless Overlap Extension PCR (OE-PCR)

In primer-less overlap extension PCR, the internal overlapping sequence of U-SS, D and Lib fragments are used as primers until full U-Lib-D fragments are assembled. A Q5-mediated PCR is performed in a volume of 25 μL in the absence of primers using 500 fmol of each fragment as template. In our experience, 15 cycles of extension are enough to fully assemble the cassette. The U-Lib-D fragment should be detectable on an agarose gel, ensuring that a large number of individual molecules have been assembled. In order to obtain high concentrations of linear donor DNA needed for transformation, the U-Lib-D products of the OE-PCR reaction are amplified in a standard Q5-mediated PCR using external primers (forward primer from pair B1 and reverse from pair D1). The expected band is purified from agarose gel using the Monarch Gel Extraction Kit or an equivalent kit prior to transformation.

Gibson Assembly

Instead of the OE-PCR, the U-Lib-D can be assembled by mixing 15 μL of Gibson Master Mix (see Note 2) with 500 fmol of each of the fragments, 0.5 μL of DpnI, and ddH2O to 20 μL. The DNA is then incubated for 1 h at 50
C. The U-Lib-D fragment should be detectable on agarose gel before further amplifying it by PCR as described in the previous section.

3.1.7 Cassette Assembly in pTarget to Generate Large Libraries

Integrating the donor cassette into the pTarget plasmid gave us more efficient genome integration, leading to a 10- to 100-fold increase in library size compared to the linear donor DNA method. However, this is at the cost of a more laborious cassette construction process. In order to clone the donor DNA into the plasmid, the homologous arms (U and D) are amplified using primer pairs B2 and D2, which includes flanking regions homologous to pTarget for Gibson assembly. 1. Amplify the pTarget-N20 backbone using primer pair E. Purify it using the Monarch PCR & DNA Cleanup kit or equivalent.

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2. Mix equimolar amounts (500 fmol) of the U, SS-pbpA (PCR product of primer pair C) and D fragments together with plasmid backbone in 15 μL of Gibson Master Mix (see Note 2), 0.5 μL of DpnI, and ddH2O to 20 μL, and incubate for 1 h at 50
C. 3. Transform 1 μL of the assembly mix into E. coli TOP10 competent cells. 4. Extract the plasmid harboring the donor DNA cassette (pTarget-N20-U-SS-pbpA-D) by plasmid miniprep. 5. Amplify the plasmid/cassette template obtained in step 4 with primer pair G to amplify the plasmid backbone without pbpA in order to assemble it with the library between U-SS and D. 6. Mix equimolar amounts (500 fmol) of the Lib fragment and plasmid backbone with 15 μL of Gibson Master Mix, 0.5 μL of DpnI, and ddH2O to 20 μL, and incubate for 1 h at 50
C. 7. Clean the Gibson product using the Monarch PCR & DNA Cleanup kit or equivalent. Elute the DNA in 7 μL of ddH2O and use 4 μL of the DNA mix for electroporating E. coli TOP10 competent cells. 8. Verify random colonies by colony PCR for the insertion of ULib-D into pTarget using primer pair. The size of the library is estimated by calculating the number of transformants obtained at step 7, and the expected library size is achieved by scaling up. 9. Scrape all colonies from plates with LB medium using a spreader. 10. Pool the bacteria and extract the plasmid library using a QIAGEN Plasmid Maxi Kit or equivalent maxi prep kit to obtain the maximum amounts of plasmids for the genome modification step. 3.1.8 Preparation of E. coli TOP10 Electrocompetent Cells Harboring pCas

1. Plate E. coli cells harboring pCas on LB agar containing kanamycin (50 mg/L) and incubate O/N at 30
C (see Note 3). 2. Among the grown colonies, pick a small one (this is very important, see Note 4) and inoculate in 10 mL of LB containing 50 mg/L of kanamycin (LB-Kan) in a 15 mL centrifuge tube and incubate overnight at 30
C without agitation (see Note 5). 3. Start a culture by inoculating 40 mL of LB-Kan with 400 μL of preculture. Incubate in a 500 mL flask at 30
C and 180 rpm until an OD600 between 0.4 and 0.6 is reached.

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4. Add arabinose (10 mM final concentration) to the culture to induce the λ-Red recombination proteins. Incubate for 15 min at 30
C and 180 rpm. 5. Chill the culture in a water–ice bath for 30 min. 6. Transfer the culture to a prechilled 50 mL centrifuge tube and centrifuge at 4600  g and 4
C for 7 min. 7. Add 30 mL of ice-cold resuspend the cells.

ddH2O

to the pellet. Swirl gently to

8. Centrifuge again at 4600  g and 4
C for 7 min. 9. Resuspend the pellet in 1 mL of chilled ddH2O. Transfer it using a 1000 μL sterile tip to a prechilled sterile 1.5 mL microcentrifuge tube. 10. Centrifuge down at 10,000  g and 4
C for 4 s in a benchtop centrifuge. 11. Carefully pipet out the supernatant and repeat this step once more. 12. Resuspend cells in 200 μL of chilled ddH2O. Keep on ice until transformation. 3.1.9 Transformation

1. Add either 100 ng of pTarget-N20-U-Lib-D or 100 ng of pTarget-N20 and 400 ng of linear donor U-Lib-D cassette to 50 μL of competent cells. 2. Electroporate the cells in a 2 mm cuvette and recover them in 950 μL of SOC medium. 3. Incubate them for 45 min at 30
C before plating all the cells on LB-Kan-Tet plates. Incubate overnight at 30
C. 4. Library size is estimated by counting the number of individual transformants.

3.1.10 Verification of Genome Modification

Random colonies are verified by colony PCR using primers outside the recombination arms (primer pair H). The validation of positive clones as well as their mutation rate (~4–6 mutation/gene in our case) is checked by sequencing of the colony PCR product using internal primer pair I. Library size is corrected if a fraction of clones does not contain the gene of interest. If the library size is not sufficient, the whole process should be repeated, upscaling the reaction as needed.

3.1.11

In order to cure pTarget, the colonies harboring our library are scraped from the plates with LB medium using a spreader and 0.1 mL is inoculated into 10 mL of LB medium supplemented with 50 mg/L of kanamycin and 0.5 mM IPTG. After incubation at 30
C overnight, 0.1 mL of the culture is diluted in 10 mL of LB supplemented with 50 mg/L of streptomycin (see Note 6) and

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incubated at 37
C overnight to cure the pCas plasmid. The curing is confirmed by the inability of cells to grow on LB-tetracycline (12.5 mg/L) or LB-kanamycin (50 mg/L). 3.2 Libraries in S. thermophilus Using Natural Competence 3.2.1 Principle

Natural transformation is the capability of several species of bacteria to achieve a transient competent state in which they are able to take up exogenous DNA inside the cells. This DNA can be used by the cells to fulfill nutritional requirements for deoxyribonucleotides, or, depending on the sequence similarity to the chromosome, it can be integrated into the chromosome by homologous recombination [22]. S. thermophilus is one of the species in which natural transformation has been well described [23]. The bacterium is not pathogenic (GRAS status, Generally Recognized As Safe). Some strains featuring very high transformation efficiencies have been reported and, combined with a highly efficient intrinsic recombinogenic activity, those strains are interesting candidates for the creation of high diversity chromosomal libraries [24]. In our hands, the method described herein has been successfully used for transforming up to 50% of the whole bacterial population, indicating that libraries containing up to ten billion individual transformants can be easily built. The mechanism of competence induction in S. thermophilus is shown in Fig. 4. Under exponential growth conditions, the expression of comS is triggered, leading to the production of the precursor (pre-ComS) of the peptide pheromone. Then, pre-ComS is exported and matured extracellularly by a protease cleavage to produce mature ComS, also called XIP (comX inducing peptide). Above a certain concentration threshold, extracellular XIP initiates peptide (re)importation by the Ami/Opp transporter into the cytoplasm. Intracellularly, it interacts with the transcriptional activator ComR, forming a dimeric ComR–XIP complex that binds to the ComR box located in the promoter of comS and comX genes to promote their transcription, and thus inducing a positive feedback loop through the induction of comS (see Fig. 4). Eventually, ComX induces transcription of late competence genes necessary for transcription of genes involved in DNA uptake and processing [13, 23, 25, 26]. Competence can be artificially induced by adding a synthetic XIP peptide (ComS17–24 LPYFAGCL) to the growth medium [23, 24]. Naturally competent bacteria offer a powerful approach to directly manipulate the chromosome of bacteria. Typical methods to modify the genome involve creating bicistronic cassettes including antibiotic resistance gene in addition to the gene of interest. However, this can be complicated when multiple mutations have to be introduced at different genomic loci, increasing the number of resistance gene required [27]. An elegant scarless strategy to circumvent the problem is based on the replacement of a negative selection marker by the gene or the library of interest.

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ComS (XIP)

pre-ComS

B. A. C.

pre-ComS ComR

comS

comR

comX

Competent state

D.

sX

Late competence genes

Fig. 4 Competence regulation by the comRS system in S. thermophilus. (a) Expression of comS initiates the system; the precursor of the peptide pheromone ComS is exported and matured. (b) On reaching a threshold of extracellular concentration of the matured peptide pheromone (XIP), it is reimported into the cell. (c) In the cytoplasm, XIP interacts with ComR. The dimeric ComR.XIP complex binds to regions upstream and activates transcription of genes comS and comX. (d) ComX is a sigma factor that induces transcription of late competence genes required for DNA transformation. (Figure modified from [26])

Our method, schematized in Fig. 5, was developed with the orotate transporter (oroP)-based selection/counterselection marker, as described previously [28, 29]. The presence of the oroP gene confers the ability to utilize orotate as the sole pyrimidine source in a pyrimidine auxotrophic strain, therefore acting as a positive selection marker. Uracil auxotrophic mutants could be selected by the method described previously [30]. Additionally, the presence of oroP results in sensitivity to 5-fluoroorotate (5-FOA). In our method, we only utilized this property by selecting for 5-FOA resistance as a consequence of oroP replacement by the library insertion (Fig. 5B). Before library construction, S. thermophilus is therefore modified by inserting a bicistronic cassette carrying oroP and the cat gene in the target locus (Fig. 5A). The cat gene encodes chloramphenicol acetyl transferase, it is expressed from the P32 constitutive promoter and confers resistance to chloramphenicol (CmR) while oroP is expressed under a synthetic promoter as described previously [28]. Another system for negative selection is based on the rpsL gene [27]. In our experience however, the rpsL system generates a high frequency of false-positive transformants.

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A.

S. thermophilus chromosome

U U

cat

U

D

U

oroP D

Select transformants with chloramphenicol

C.

B. cat

lib

oroP D

D

Select transformants with 5-FOA

Start culture at OD600 0.05 in milk, incubate at 37C for 75 minutes

D. XIP

Add XIP at a final concentration of 1µM and donor DNA Incubate at 37C for 3 hours !"#

E.

F.

Plate on M17L with chloramphenicol/5-fluoroorotate

Incubate overnight at 37C in an anaerobic jar

Fig. 5 Overview of the protocol designed for creating chromosomal libraries using natural transformation in S. thermophilus. (a) A cat-oroP chimeric cassette with upstream (U-arm) and downstream (D-arm) homology fragments was constructed in vitro by Gibson assembly. The cassette was introduced in the chromosome by natural transformation and double homologous recombination. Transformants were selected for acquired resistance to chloramphenicol. (b) The mutant strain from step A was transformed with the ep-PCR library assembled with U- and D-arms as well. Transformants were selected for loss of oroP fragment, resulting in resistance to 5-FOA. (c–f) Schematic protocol for genetic modification using natural transformation in S. thermophilus

As an example, the following protocols are described for building a library of leuB genes (1038 bp) created by error-prone PCR and inserted into the tRNASer locus of S. thermophilus. To achieve this, a recipient strain containing a cat-oroP cassette inserted at the target locus is built. In a second step, the cassette is replaced by the library of genes using the counterselection principle.

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A cassette comprising cat and oroP genes under constitutive promoter control and flanked by 1.5-kb DNA fragments corresponding to the upstream (U) and downstream (D) regions of the tRNASer locus is assembled (see Note 7). All primers used in this section can be found in Table 1 and are designed with 40 nucleotides overlapping sequences. 1. Primer pairs K1 and K2 are used to amplify the U and D fragments from S. thermophilus genomic DNA as template, respectively (see Note 8). 2. Primer pairs K3 is used to amplify the cat fragment from the strain described in [25]. 3. Primer pair K4 is used to amplify the oroP fragment from plasmid pCS1966 described in [28]. 4. Clean up the fragments by using the Monarch PCR & DNA Cleanup kit or equivalent. 5. Mix the four purified DNA fragments to get equimolar (1 pmol) concentration, add ddH2O to 5 μL (see Note 9). 6. Incubate with 15 μL of the Gibson Master Mix (see Note 10) for 1 h at 50
C. 7. Verify the presence of the expected band of full-length cassette on agarose gel. 8. Clean up the product using column purification. Elute in ~10 μL of ddH2O or elution buffer.

3.2.3 Chromosomal Insertion of U-cat-oroP-D Cassette at the tRNASer Locus (Adapted from [25])

Day 0 1. Start a preculture of S. thermophilus LMD9 in 1 mL of CDML (see Note 11) at 37
C without agitation. Day 1 1. Dilute the culture in 1 mL of skimmed milk medium (SMM) to an OD600 of 0.05 (typically 50 μL in 1 mL milk) and incubate for 75 min at 37
C without agitation (see Note 12).

2. Thaw XIP peptide (500 μM stock) and add 2 μL to the culture (1 μM final). 3. Mix 300 μL of cells in a 1.5 mL microcentrifuge tube with the purified assembled donor DNA cassette (10 μL) from Subheading 3.2.2. Take 300 μL in another microcentrifuge tube as control (without donor DNA). 4. Incubate both tubes at 37
C for 3 h without agitation. 5. Spread 100 μL of cells on M17L + chloramphenicol plates. 6. Incubate the plates O/N at 37
C in an anaerobic jar.

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Day 2 1. Pick several clones and restreak them on M17L + chloramphenicol to prevent that nonresistant cells propagate in association with resistant cells (streptococci can form chains) (see Note 13).

2. Incubate the plate overnight at 37
C in an anaerobic jar. Day 3 1. Pick an isolated colony of each clone, perform colony PCR (see Subheading 3.3 for detailed protocol) to verify presence of insert and cultivate one clone to store it as a glycerol stock (Day 4). 3.2.4 Assembling the U-lib-D Cassette

A cassette comprising a library of leuB genes under its constitutive promoter and flanked by 1.5-kb DNA fragments corresponding to the upstream (U) and downstream (D) regions of the tRNASer locus is assembled. All primers used in this section are in Table 1 and were designed with 40 nucleotides overlapping sequences. 1. Primer pairs K5 and K6 are used to amplify the U and D fragments, respectively. 2. Primer pair K7 is used to perform an error-prone PCR on the leuB gene as described in Subheading 3.3. The product is called lib. 3. Clean up the PCR products with using the Monarch PCR & DNA Cleanup kit or equivalent. 4. Mix the three purified DNA fragments to get equimolar (1 pmol) concentration, add ddH2O to 5 μL (see Note 9). 5. Add 15 μL of Gibson Assembly Master Mix and 20 μL. Incubate for 1 h at 50
C.

ddH2O

to

6. Verify the presence of the expected band of full-length cassette on agarose gel. 7. Clean up the product using the Monarch PCR & DNA Cleanup kit or equivalent. Elute in 6 μL of ddH2O or elution buffer. 8. In order to obtain large variant libraries, it is necessary to prepare large amounts of linear donor DNA. To do so, the product of Gibson assembly is further amplified by a Q5-mediated PCR using primer pair K7 (see Note 14). 3.2.5 Chromosomal Insertion of U-lib-D Library at the tRNASer Locus (Fig. 5)

Day 0 1. Start a preculture of the strain tRNASer::cat-oroP obtained in Subheading 3.2.3 in 1 mL of CDML supplemented with chloramphenicol (5 mg/L).

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Day 1 1. Dilute the culture in 1 mL of milk to an OD600 of 0.05 and incubate for 75 min at 37
C without agitation (see Note 12).

2. Thaw the XIP peptide (500 μM stock) and add 2 μL to the culture (1 μM final). 3. In a 1.5 mL microcentrifuge tube, mix 300 μL of cells with ~400 ng (typically higher concentrations lead to higher number of transformants) of the assembled U-lib-D fragment. Take 300 μL in another microcentrifuge tube as control (without donor DNA). 4. Incubate both tubes at 37
C for 3 h without agitation. 5. Spread the cells on M17L + 5-FOA (500 mg/L) plates. 6. Incubate the plates overnight at 37
C in an anaerobic jar. Day 2 1. Add 2 mL of CDML and scrape off the cells using a spreader.

2. Store the library with 30% glycerol at 80
C for further analysis. 3.3

PCR Protocols

The following three protocols are used referred to in this chapter. Q5 PCR: 10 μL of 5 Q5 Reaction Buffer, 0.7 μL of 20 mM dNTPs, 2.5 μL of 10 μM Forward and reverse primers, less than 100 ng of template DNA, 1 U Q5 High-Fidelity DNA Polymerase (NEB), and nuclease-free H2O up to 50 μL. PCR thermocycler conditions: initial denaturation at 98
C for 30 s; 25–30 cycles containing: denaturation at 98
C for 20 s; annealing at temperature calculated for each pair of primers on https://tmcalculator.neb.com/#!/main for 30 s; 20–30 s/kb extension at 72
C; a final extension for 5 min at 72
C and hold at 4
C. Error-prone PCR (ep-PCR) using GeneMorph II Random Mutagenesis Kit: 5 μL of 10 Mutazyme II reaction buffer, 1 μL of 40 mM dNTP mix (provided in kit), 0.5 μL of primer mix (250 ng/μL of each primer), 2.5 U of Mutazyme II DNA polymerase, 100 ng of template DNA (see Note 15), and nucleasefree H2O up to 50 μL. PCR thermocycler conditions: initial denaturation at 95
C for 2 min; 20–25 cycles (depending on desired mutation rate, see Note 15): denaturation at 95
C for 30 s; annealing 30 s at T ¼ Tm – 5
C (Tm calculated for each pair of primers on https://tmcalculator.neb. com/#!/main); extension 1 min (1-kb targets) or 1 min/kb (>1kb targets) at 72
C; a final extension for 10 min at 72
C and hold at 4
C.

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Optimization of mutation rate: To generate low mutation rate (between 0 and 5 mutation/kb), instructions of the GeneMorph II Random Mutagenesis kit are followed according to manufacturer. According to the kit’s protocols, mutation rates can be lowered by using higher template DNA amounts and by decreasing the number of cycles of ep-PCR reaction. Therefore, for a 1 kb fragment, an initial target amount of 100 ng is required for a run with 15–20 cycles of PCR, generating a low mutation rate between 0 and 5 mutation/kb. Colony PCR: 5 μL of 5 GoTaq Flexi Buffer, 4 μL of 25 mM MgCl2, 0.7 μL of 20 mM dNTPs, 1.5 μL of 10 μM Forward and reverse primers, 1.25 U GoTaq DNA Polymerase (Promega), and nuclease-free H2O up to 50 μL. Use a toothpick to stab a single colony and rub it inside the PCR tube to detach some cells as template. PCR thermocycler conditions: initial denaturation at 95
C for 4 min (see Note 16); 25 cycles: denaturation at 95
C for 30 s; annealing at Ta (calculated for each pair of primers on https:// tmcalculator.neb.com/#!/main) for 30 s; 1 min/kb extension at 72
C; a final extension for 7 min at 72
C and hold at 4
C.

4

Notes 1. The expression of a toxic protein from the donor DNA cassette prior to homologous recombination could prevent insertion or generate biases in the library. To minimize this bias, a strategy is to avoid putting promoter sequence in the donor DNA. However, the recombination requires that the cassette includes the region immediately upstream of the library, where the promoter is typically located. A solution is to insert our gene of interest in a locus with a known expression pattern, but far enough from the promoter so as to have room for the upstream homology arm between the promoter and the gene. For example, the library can be inserted as the second gene in a polycistronic operon controlled by a single promoter with an upstream homology arm corresponding to the first wild-type gene of the operon. In this chapter, the example in E. coli is the cloning of a pbpA library in the rhamnose operon controlled by the Prha promoter (Fig. 3). It is also important to avoid the use of the Plac and ParaBAD promoters for the library since these promoters are already used in the CRISPR-Cas9/λ-Red system. 2. We tried commercial Gibson Mix such as the NEBuilder® HiFi DNA Assembly Master Mix from New England Biolabs but observed an efficiency similar to our own Gibson Master Mix

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[10]. Given the much lower cost of our Gibson mix per transformation, we usually use it. 3. Since pCas has a thermosensitive replication origin, it is important to grow cells containing pCas at 30
C instead of 37
C. 4. Colonies of E. coli harboring pCas are heterogeneous in size. This is due to a slight toxicity of Cas9 endonuclease. It is therefore very important to start experiments with a small colony since the large ones do not express active Cas9. 5. Starting the preculture of E. coli in 10 mL of LB medium in a nonshaking 15 mL centrifuge tubes leads to a shorter lag phase when the culture is inoculated the following day. Hypothesis is that the low oxygen availability reduces cell death [31]. 6. In order to prevent possible contamination, streptomycin is used after the curing steps since E. coli TOP10 cells harbors a chromosomal resistance gene. 7. Expression levels of genes inserted in different chromosomal loci vary significantly. Care should be taken when choosing an insertion site in the chromosome. Known neutral loci such as that of tRNASer are recommended. 8. The length of the homology arms is important for the efficiency of transformation in S. thermophilus. We have found that a minimum length of 800 bp for each of the flanking homologous arm is required. Transformation efficiency improves until 1500 bp where efficiency reaches a plateau. 9. The NEB Biocalculator (https://nebiocalculator.neb.com/) is a handy tool to calculate several parameters of DNA oligonucleotides and fragments such as molarity and molecular weights. 10. Alternatively, overlap extension PCR (OE-PCR) can be used to assemble DNA fragments with sufficient homology ( 4000 TON, 96% ee 4 M = Ru, Ar = p-cymene, up to 100 TON, 94% ee 5 M = Ru, Ar = p-cymene, 94 TON, 52% ee 6 M = Ru, Ar = C6H6, upt to 100 TON, 92% ee

O

O HN H

NH H O

NH H

H N

S

Cl

tBu

H N

S

P Pd

O

M Cl

tBu

Cl

2

8 M = Rh, C-H activation, up to 95 TON, 82% ee 9 M = Ir, transfer hydrogenation, up to 961 TON

10 Suzuki coupling for biaryls up to 160 TON, 90% ee

O HN H

Ph P

O

O

HN H

NH H

1 hydrogenation up to 104 TON, 96% ee

HN H

217

O N

Ru

NH

O

N

H N

S

OH2

HN H

NH O

S

O 11 deallylation, up to 150 TON

F

H O

O 12 reagent for biotinylation

F

F

F F

Fig. 2 A selection of biotinylated cofactor precursors, employed with Sav for the formation of active artificial metalloenzymes and some benchmark performance values. The color of the metal centers corresponds to Fig. 1. COD ¼ 1,5-cyclooctadiene. References: 1 [21], 2 [22], 3 [18], 4–6 [14], 7 [17], 8 [23], 9 [18], 10 [16], 11 [19], 12 [24]

The structures depicted in Fig. 2 describe either isolated complexes [3–10] or are likely species formed in situ when reacting the biotinylated ligand with a commercial organometallic catalyst precursor [1, 2, 11] prior to addition of streptavidin. The precursors

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shown are not necessarily part of the catalytic cycle of the actual catalyst. They, however, give rise to active artificial metalloenzymes when treated with streptavidin as described above and being subjected to the reaction conditions and substrates. Biotinylated ligand 1 [21], originally reported by Whitesides [26], and ligand 2 [22], contain first coordination sphere features of classical bidentate diphosphine ligands, most prominently employed in asymmetric hydrogenation. They carry two bulky substituents at phosphorous, namely, phenyl groups, which define the spatial organization around the metal center by blocking “quadrants” for substrate approach and orientation [27]. The conformation of the diphosphine ligand is here controlled by the protein scaffold—arbitrary conformations are shown in Fig. 2—and not by the chirality of the backbone as is common in small-molecule catalysts for asymmetric synthesis. Complexes 3–6 [14, 18] constitute a biotinylated version of Noyori’s ruthenium-TsDPEN complexes [28] and their iridium and rhodium analogues published by Mashima and Tani [29]. These complexes contain an asymmetrically substituted metal center, which is believed to be responsible for asymmetric induction in the catalytic conversion of prochiral substrates. While the absolute configuration at the metal center is in Noyori’s catalyst controlled by substituents at the ethylene bridge between the ligating nitrogen atoms, here the protein scaffold favors one configuration over the other [20]. Cofactor 7 [17] is a biotinylated version of the Hoyveda-Grubbs second generation catalyst [30, 31] and has been employed in a directed evolution study of artificial metalloenzymes. An alternative attachment position of the biotin anchor has also been investigated [32]. In complexes 8 and 9, biotin is linked to the aromatic cap of the piano-stool complex in contrast to complexes 3–6, leaving three coordination sites available. While these are required for the investigated C-H activation reaction with the rhodium complex 8 [23], in transfer hydrogenation two coordination sites can be occupied by commercial bidentate ligands, thereby giving rapid access to diversity of biotinylated cofactors for transfer hydrogenation [33, 34]. Complex 10 is one of the few complexes investigated where a non-chelating biotinylated ligand ensures incorporation into streptavidin [16]; another important example is complex 7. Catalytic results and the X-ray structure for 10@Sav indicated, however, that the metal center is reliably localized in the biotin-binding-site-vestibule. Complex 11 has been probed in directed evolution protocols with surfacedisplayed streptavidin constructs [19]. Once suitable cofactor and protein variant have been identified, the large-scale expression of the streptavidin mutant can be undertaken and is described in the following in the form of a step-by-step protocol.

Streptavidin-Based Artificial Metalloenzymes

1.2 Large-Scale Production, Purification, and Analysis of Streptavidin

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As mentioned above, streptavidin is a naturally occurring protein in the bacterium Streptomyces avidinii [35]. Since Sav has a bacterial origin and thus does not undergo any complex posttranslational modification, Escherichia coli (E. coli) is an ideal strain for heterologous protein production [36]. By taking advantage of the T7 RNA polymerase/T7 promoter expression system [37], the recombinant Sav gene is transcribed in the E. coli strain BL21(DE3) by a T7 RNA polymerase, which itself is encoded on the bacterial genome and regulated by a lacUV5 promotor. The T7 RNA polymerase is therefore expressed upon the addition of inducer molecules like isopropyl β-D-1-thiogalactopyranoside (IPTG) or lactose [38]. In our expression system, mature Sav is preferred over core Sav, which is mostly expressed as insoluble protein [8] and accordingly less suitable for convenient protein production. The DNA sequence for the mature 159 amino acid long Sav monomer is localized on the expression vector pET-11b under control of a T7 promoter [39]. Only residues 15–159, however, belong to the natural Sav sequence, whereas the first N-terminal amino acids are replaced by a T7-tag peptide (residue 1–11) followed by Arg, Asp, and Gln (Fig. 3). The T7-tag peptide is hypothesized to improve the solubility of the translated Sav gene [42]. The construct will be called from now on T7Sav. T7Sav is typically expressed in soluble form. The cell-free extracts are subsequently frequently dialyzed against 6 M guanidinium chloride (GdmCl) [8, 43]. To generate a variety of T7Sav mutants, the T7Sav gene can be subjected to sitedirected mutagenesis (SDM), following the QuikChange protocol from Stratagene [15].

Fig. 3 DNA and protein sequence of the T7-tagged mature streptavidin (T7Sav) construct that is encoded on the expression vector pET-11b and used for protein overexpression. The N-terminal methionine residue is cleaved off following translation [39]. Amino acid residues 13–139 (with Ala and Glu at positions 13 and 14, respectively) correspond to natural core-streptavidin [40, 41]

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Fig. 4 SDS-PAGE result after cell lysis of E. coli cultures, which were grown for 24 h at 30  C in the autoinduction medium ZYP-5052 [38] in order to produce the target protein T7Sav. Over-expressed tetrameric T7Sav (S112E is the variant depicted here) can be seen under UV-light by the detection of the bound fluorophore B4F (a), and after protein staining with Coomassie brilliant blue R (b) between 50 and 60 kDa. The vast majority of over-expressed T7Sav is found in the soluble fraction. Purified T7Sav WT was loaded as a positive control. (c) Deconvoluted ESI-MS spectrum of monomeric T7Sav; Instrument: MS Bruker Daltonics microTOF (API). The variant K121Y (calculated mass of the monomer: 16459.9 Da) is reliably detected at 16461.1 Da

The overexpression of T7Sav can be realized in good yields (typically 200–300 mg/L) by using the auto-inducing medium ZYP-5052 [38]. Significantly higher yields have been realized with a fed-batch procedure [44]. The main advantage of this rich medium is that all required compounds for protein production are already present from the beginning of cell growth. Thus, manual addition of inducer molecules as well as regular measurements of the optical density (OD) to determine the ideal time for induction can be omitted (see Note 1). Cell growth in our approach is carried out in a 30 L fermentor for 24 h at 30  C under steady growth conditions, i.e., stable pH and constant aeration. Under these conditions, OD600nm values of 10–15 are typically reached after 24 h of cell growth (see Note 2). Using the fluorophore biotin-4fluorescein (B4F), successful expression of T7Sav as a soluble and functionally active tetrameric protein can be detected by SDS-PAGE [39]. The molecular mass of purified and lyophilized T7Sav variants is finally reliably confirmed by electrospray ionization mass spectrometry (ESI-MS, Fig. 4).

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Fig. 5 B4F binding assay result for the detection of FBS in tetrameric T7Sav. Protein samples were measured in triplicates. Average values of all three samples were plotted, and standard deviations indicate measurement errors. (a) Data points showing a low fluorescence signal (red rectangles) indicate quenching of the B4F fluorescence due to binding to T7Sav. As soon as all four binding sites in the tetramer are saturated by B4F (usually at four equivalents), fluorescence increases at a higher slope with the addition of B4F (blue rectangles). Linear regression curves can be used to fit values with low and values with high fluorescence. (b) Calculation of the intersection between the straight lines reveals the number of FBS. For the T7Sav WT variant, which is depicted here, the value of FBS was determined to be 3.9 (see Note 25)

By implementing a binding assay, which detects B4F, the number of free binding sites (FBS) per T7Sav tetramer can be determined [45]. The assay is based on the concept that binding of B4F to Sav results in quenching of ligand fluorescence. Saturation of all binding sites upon continuous addition of B4F results in a sudden increase in fluorescence intensity. The assay reveals that purified T7Sav tetramers are in general able to bind 3.5–4 molecules of biotin (Fig. 5).

2

Materials

2.1 Plasmids and Cells

The expression vector pET-11b (Novagen) containing the cDNA of the mature Sav (residues 15–159) and the N-terminal T7 tag peptide (11 residues followed by Arg, Asp, and Gln) was a gift from Professor Paolo Santambrogio, University of Milan). The E. coli strain BL21(DE3) was used. Genotype: B F dcm  ompT hsdS(r B mB ) gal λ(DE3).

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2.2 Media and Buffers for Cell Growth and Cell Lysis, Dialysis, and Protein Purification

All solutions are prepared either in deionized water (DI-H2O) or in ultrapure water that has a resistivity of 18.2 MΩ cm (Milli-Q water). All buffers and solutions required for dialysis are stored at 4  C. For filter sterilization, 0.22 μm pore-sized syringe filters are used. Cell-free extracts (CFE) and buffers for protein purification are all filter-sterilized and degassed using 0.2 μm pore-sized bottle top filters and an ultrasound bath, respectively. Ampicillin stock solution (100 mg/mL): 2 g of ampicillin sodium salt is solubilized in 20 mL DI-H2O, is filter sterilized for a 20-L-fermentation, and can be stored as 1 mL aliquots at 20  C for up to a year. Lysogeny Broth (LB) medium: 10 g of tryptone (1% w/v), 5 g of yeast extract (0.5% w/v), and 5 g of NaCl (0.5% w/v) are dissolved in DI-H2O (final volume of 1 L), autoclaved (20 min at 121  C, 1.5 bar), and stored at room temperature (RT) for up to 4 months. Prior to use, the ampicillin stock solution is added to a final concentration of 100 μg/mL. LB agar plates: The recipe is analogue to the one for LB medium (see above) with the following modifications. In addition to tryptone, yeast extract, and NaCl, 15 g of agar (1.5% w/v) is added to 1 L medium. The solution is autoclaved (20 min, 121  C, 1.5 bar) and chilled to 55  C prior to addition of the ampicillin stock solution to a final concentration of 100 μg/mL (see Note 3). The liquid medium is subsequently poured into Petri dishes. Plates with solidified LB agar are stored at 4  C for up to 4 months. 20 ZYP salts: 136 g of KH2PO4 (50 mM), 142 g of Na2HPO4 (50 mM), and 66 g of (NH4)2SO4 (25 mM) are dissolved in DI-H2O (final volume of 1 L) and autoclaved (20 min, 121  C, 1.5 bar). The amount is sufficient for one 20-L-fermentation (see Note 4). 20 ZYP sugars: 100 g of glycerol (10% v/v), 11 g of glucosemonohydrate (1.1% w/v), and 40 g of α-lactose monohydrate (4% w/v) are dissolved in DI-H2O (final volume of 1 L) and autoclaved (20 min, 121  C, 1.5 bar). The amount is sufficient for one 20-Lfermentation (see Note 4). 200 mM MgSO4: 4.8 g of anhydrous MgSO4 is dissolved in DI-H2O (final volume of 200 mL) and autoclaved (20 min, 121  C, 1.5 bar). The amount is sufficient for one 20-L-fermentation (see Note 4). 200 mM MgCl2 stock solution: 3.81 g of MgCl2 is dissolved in DI-H2O (final volume of 200 mL) and autoclaved (20 min, 121  C, 1.5 bar). Antifoam solution (1 L): 100 mL of antifoam 204 (10% v/v) is mixed with 900 mL of DI-H2O. The solution is autoclaved (20 min, 121  C, 1.5 bar) and stored at RT for up to 6 months (see Note 5). Auto-induction (ZYP-5052) medium: For a 20 L fermentor culture, 200 g of tryptone (1% w/v) and 100 g of yeast extract (0.5% w/v) are dissolved in 17.8 L of DI-H2O. The solution is

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filled into the fermentor vessel and autoclaved in situ (30 min, 121  C). The medium is chilled to 55  C or lower before the sterilized solutions (1 L of 20 ZYP salts, 1 L of 20 ZYP sugars, and 200 mL of 200 mM MgSO4) are added to the vessel. The ampicillin stock solution is added to a final concentration of 100 μg/mL. LBSOB medium: For 1 L of medium 20 g of tryptone (final concentration 2% w/v), 5 g of yeast extract (final concentration 0.5% w/v), 0.584 g of NaCl (final concentration 10 mM), and 0.186 g of KCl (final concentration 2.5 mM) are dissolved in 900 mL of DI-H2O and autoclaved (20 min at 121  C, 1.5 bar). The medium can be stored at RT for up to 4 months. To 90 mL of medium, 5 mL of 200 mM MgCl2 (final concentration 10 mM) and 5 mL of the 200 mM MgSO4 (final concentration 10 mM) stock solution are added (see Note 6). RF1 buffer: 2.42 g of RbCl (final concentration 100 mM), 1.98 g of MnCl2·4H2O (final concentration 50 mM), 0.59 g of KOAc (final concentration 30 mM), 0.29 g of CaCl2·2H2O (final concentration 10 mM), and 30 mL of glycerol (final concentration 15% v/v) are dissolved in Milli-Q water (final volume of 200 mL). The pH is adjusted to 5.8 with 0.2 M CH3COOH. The buffer is filter-sterilized and can be stored at 4  C for up to 6 months. RF2 buffer: 0.05 g of RbCl (final concentration 10 mM), 0.44 g of CaCl2·2H2O (75 mM), 0.084 g of MOPS (10 mM), and 6 mL of glycerol (15% v/v) are dissolved in Milli-Q water (final volume of 40 mL). The pH is adjusted to 7.0 with 1 M NaOH. The buffer is filter-sterilized and stored at 4  C for up to 6 months. Lysis buffer: To make 1.5 L of a 20 mM Tris–HCl, pH 7.4 buffer, 30 mL of 1 M Tris–HCl, pH 7.4 is diluted to a final volume of 1.5 L with DI-H2O. The buffer is thoroughly mixed with 1.5 g of lysozyme (1 mg/mL) and 3 mg of DNaseI (2 μg/mL) (see Note 7). 6 M guanidinium chloride (GdmCl) dialysis buffer, pH 1.5: 8.6 kg of GdmCl is dissolved in DI-H2O. To adjust the pH to 1.5, 37% HCl is added. DI-H2O is added to a total volume of 15 L. The buffer can be kept at 4  C for up to 6 months (see Note 8). 20 mM (10 mM) Tris–HCl dialysis buffer, pH 7.4: 300 mL (150 mL) of 1 M Tris–HCl, pH 7.4 is diluted to a final volume of 15 L with DI-H2O and kept at 4  C (see Note 9). Iminobiotin binding (IBB) dialysis buffer, pH 10.8: 63 g of NaHCO3 (final concentration 50 mM) and 438 g of NaCl (final concentration 0.5 M) are dissolved in DI-H2O. The pH is adjusted to 10.8 with 5 M NaOH before DI-H2O is added to a total volume of 15 L and stored at 4  C (see Note 9). Iminobiotin binding (IBB) buffer, pH 10.8: 8.4 g of NaHCO3 (50 mM) and 58.4 g of NaCl (0.5 M) are dissolved in Milli-Q water. The pH is adjusted to 10.8 with 5 M NaOH before

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Milli-Q water is added to a total volume of 2 L. The buffer is filtered and stored at 4  C for up to 6 months (see Note 10). Elution buffer: 20 mL of glacial acetic acid (1% v/v) is mixed with Milli-Q water (final volume of 2 L). The buffer is filtered and stored at 4  C for up to 6 months. 2.3 Solutions for the Detection of Free Binding Sites (FBS) in T7Sav Tetramers

Monobasic stock solution: 15.6 g of NaH2PO4·2H2O (final concentration 200 mM) is dissolved in 500 mL of DI-H2O and autoclaved (20 min, 121  C, 1.5 bar). The solution can be stored at RT for several weeks. Dibasic stock solution: 53.65 g of Na2HPO4·7H2O (final concentration 200 mM) is dissolved in 1 L of DI-H2O and autoclaved (20 min, 121  C, 1.5 bar). The solution can be stored at RT for several weeks. 0.1 M phosphate buffer with BSA, pH 7.0: 58.5 mL of monobasic stock solution is mixed with 91.5 mL of dibasic stock solution and made up to 300 mL with Milli-Q water. The buffer is filter-sterilized and kept for up to 1 month at RT. Prior to use, bovine serum albumin (BSA) is added to the required amount of buffer to a final concentration of 0.1 mg/mL. 100 μM protein stock solution: To make this solution, the molecular weight (MW) of the tetramer is calculated for each T7Sav variant using the ProtParam online tool from ExPASy (https:// web.expasy.org/protparam/). Each T7Sav variant is analyzed in triplicate. For this purpose, three 100 μM protein stock solutions are prepared using Milli-Q water (see Note 11). B4F stock solution: To make a 0.6 mM biotin-4-fluorescein (B4F) solution, 10 mg of B4F (CAS: 1032732–74-3, MW: 644.7 g/mol) are dissolved in 25.85 mL of DMSO. One milliliter aliquots of this solution are stored for several months at 80  C (see Note 12).

2.4 Polyacrylamide Gels and Buffers for SDS-PAGE

Production of the target protein T7Sav is verified after 24 h of cultivation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini-PROTEAN Tetra Cell system from Bio-Rad. Since T7Sav does not contain any disulfide bridges, reducing agents such as DTT are not added to the loading dye. In order to preserve the quaternary structure of T7Sav, the protein solutions are not heated up to high temperatures prior to electrophoretic separation [39]. The fluorophore B4F is added to reveal functional tetrameric T7Sav in soluble and insoluble fractions. Separating gel, 12% acrylamide gel: 2.5 mL of Milli-Q water, 3 mL of 30% (v/v) acrylamide 4K solution (Applichem, cat. no. A1672), 3.8 mL of 1.5 M Tris–HCl, pH 8.8, 75 μL of 20% (w/v) SDS solution, 100 μL of 15% (w/v) ammonium persulfate, and 3 μL of N,N,N0 ,N0 -tetramethyl-ethylenediamine (TEMED) are thoroughly mixed. The solution is cast between two glass plates

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held apart from each other by a 1.5 mm spacer. On top of the mixture, a layer of Milli-Q water is added. As soon as the separating gel has become solid, the water is removed. Stacking gel, 5% acrylamide gel: 1.7 mL of Milli-Q water, 500 μL of 30% (v/v) acrylamide, 750 μL of 0.5 M Tris–HCl, pH 6.8, 15 μL of 20% (w/v) SDS solution, 20 μL of 15% (w/v) APS, and 3 μL of TEMED are vigorously mixed before being poured on top of the separating gel. A 15-well comb is inserted into the gel on top. 5 SDS running buffer: 30.2 g of Tris-base, 144 g of glycine, and 10 g of SDS are weighed in and made up to 2 L by the addition of DI-H2O. The buffer is stored at RT. Prior to use, the buffer is diluted to 1 SDS running buffer. 3 SDS loading dye: 2.4 mL of 1 M Tris–HCl, pH 6.8, 6 mg of bromo-phenol blue, 3 mL of glycerol, and 3 mL of 20% (w/v) SDS solution are made up to 10 mL with Milli-Q water. The dye solution can be stored at RT for up to 12 months. T7Sav wild-type (WT) stock solution: To 1 mg T7Sav WT, 1 mL of 50% (v/v) glycerol dissolved in Milli-Q water is added. The solution is stored at 20  C. Protein staining solution: 0.5 g of Coomassie Brilliant blue R, 1 L of EtOH, and 150 mL of glacial acetic acid are mixed and made up to 2 L with DI-H2O. The solution is kept at RT. Protein destaining solution: 400 mL of EtOH and 200 mL of glacial acetic acid are mixed and made up to 2 L with DI-H2O. The solution is kept at RT.

3

Methods

3.1 Preparation of Chemically Competent BL21(DE3) Cells

Required solutions, media, and techniques are described by Hanahan and colleagues [46, 47]. Buffers and media, as well as devices that get in direct contact with cells, need to be sterilized. Moreover, as soon as cells are harvested, it is strictly required to maintain all cell-containing solutions, as well as devices, e.g., centrifuges and rotors, at 4  C. To avoid contamination, the following steps are conducted in a laminar flow hood (see Note 13). 1. An overnight culture is prepared by suspending a small amount of frozen cells from a commercially available glycerol stock, i.e., BL21(DE3) in 5 mL of LB medium. The cell culture is incubated overnight at 200 rpm (stroke ¼ 1 in.) and 37  C. 2. The cell suspensions are diluted to an OD600nm of 0.04 in 100 mL of LBSOB medium. Growth is continued at 200 rpm (stroke ¼ 1 in.) and 37  C until an OD600nm of 0.32–0.35 is reached (see Note 14).

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3. The cell suspension is centrifuged (4  C, 4000  g, 10 min); cells are resuspended in 20 mL ice-cold RF1 buffer and incubated on ice for 15 min. 4. Cells are again separated by centrifugation (4  C, 4000  g, 10 min), resuspended in 4 mL ice-cold RF2 buffer, and incubated on ice for another 15 min. 5. The suspension is split into 50 μL aliquots, which are shockfrozen in liquid nitrogen and then stored for several months at 80  C. 3.2 Plasmid Transformation into Chemically Competent BL21(DE3) Cells

The work flow for primer design and SDM for pET expression vectors harboring the natural T7Sav gene are well described by Mallin et al. [15]. 1. A 50 μL aliquot of chemically competent BL21(DE3) cells is thawed on ice for 10 min. 2. 0.5–1 μL of plasmid DNA (approximately 100–200 ng) is carefully added to the cells. The suspension is incubated on ice for up to 20 min. 3. The cells are subjected to a heat-shock (42  C, 30 s) and then again incubated on ice for 2 min. 4. 500 μL of LB or LBSOB is added to the cells. Incubation for up to 1 h at 37  C is allowed (see Note 15). 5. Cells are quickly spun down (20,000  g, 15 s) and resuspended in 150 μL of supernatant (the remaining supernatant is discarded). 6. The cell suspension is streaked onto a LB agar plate containing 100 μg/mL of ampicillin. The plate is incubated overnight at 37  C and can then be stored for up to 1 month at 4  C when sealed with a parafilm stripe. 7. The next day, a single colony is picked and used for an overnight culture.

3.3 Large-Scale Overexpression of Recombinant T7Sav by Using a 30 L Fermentor

Approaches to successfully express and purify soluble and functional T7Sav in high yields in E. coli have been previously described by us [39, 48]. In this overview, we take up most of the important aspects but describe, however, some modifications in order to optimize or simplify the entire procedure. Whereas most of or T7Sav variants that were generated by SDM express as soluble, properly folded, and functional proteins in good yields, we often observe either low production levels or the formation of inclusion bodies [14]. Misfolded T7Sav constructs, which tend to form inclusion bodies, can be subjected to refolding measures prior to affinity purification [49].

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1. The day before an overnight culture is prepared. Therefore, a 50 mL Falcon tube filled with 10 mL of LB medium and the respective amount of ampicillin (final concentration: 100 μg/ mL) is inoculated with a single colony from freshly transformed BL21(DE3) cells (see Note 16) and incubated overnight (37  C, 200 rpm, stroke ¼ 1 in.). 2. In parallel, all fermentor inlet tubings (for all added compounds) are autoclaved (20 min, 121  C, 1.5 bar). 3. The vessel and the air outlet of the fermentor are sterilized prior to use. To do this, the fermentor vessel is filled with DI-H2O to 20 L and heated up to 121  C. As soon as the entire vessel is under pressure, hot steam is released for approximately 15 min through the air outlet valve to make sure this line is thoroughly sterilized and not contaminated from previous fermentations. The fermentor vessel is cooled down to below 100  C, pressure is released, and water is removed. 4. In the next step, 20 L of auto-induction medium is prepared in the fermentor vessel as described above. 5. Prior to inoculation of the auto-induction (ZYP-5052) medium with 10 mL of overnight culture, 50–100 mL of antifoam solution is added. 6. Cell growth is permitted for 24 h under constant conditions (30  C, pH 7.0, agitation speed of 600 rpm and constant aeration). 7. Cells are harvested the next day by centrifugation (5000  g, 10 min, 4  C). The supernatant is discarded and the cell pellet is stored at 20  C. 8. The cell pellet is thawed using a warm water bath. 9. Cells are lysed in 1.5 L of lysis buffer under vigorous shaking for approximately 2 h at RT (see Note 17). 10. The cell extract (CE) is subjected to another cycle of freezing and thawing, which is supposed to improve the efficiency of cell lysis. 11. The CE is filled into dialysis bags. The first dialysis step is performed for no longer than 24 h in 6 M GdmCl dialysis buffer (see Note 18), followed by at least 8 h in 20 mM Tris– HCl dialysis buffer and finally by incubation for at least 14 h in IBB dialysis buffer (see Note 7). The CE is subjected to highspeed centrifugation (20,000  g, 50 min, 4  C). The cell pellet is discarded, whereas the cell free extract (CFE) is kept, degassed, and filtered (0.22 μm pore-size) prior to affinity chromatography.

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3.4 Detection of Over-Expressed T7Sav by SDS-PAGE

12. After 24 h of cultivation, the OD600 nm of E. coli cells in the auto-induction medium is determined at a wavelength of 600 nm. 13. 1 mL of the E. coli cell culture, normalized to an OD600 nm value of 15, is centrifuged (20,000  g, 30 s). The cell pellet is frozen, thawed, and finally resuspended in 1 mL of lysis buffer (see Note 19). 14. Cell lysis is permitted for 30 min at 37  C. The CE is spun down (20,000  g, 5 min). 15. The CFE (supernatant ¼ soluble fraction, S) is collected in a separate tube. 16. The cell pellet is solubilized under vigorous shaking in 1 mL of 8 M urea (insoluble fraction, IS). 17. 20 μL of each fraction is mixed with 1 μL of B4F stock solution and 10 μL 3 SDS loading dye. 18. To make the positive control solution, 3 μL of the T7Sav WT stock solution is diluted in 17 μL of Milli-Q water. 1 μL B4F stock solution and 10 μL 3 SDS loading dye are added. 19. 20 μL of each prepared sample (S, IS, and positive control) is filled into the wells of the prepared 12% acrylamide gel and subjected to SDS-PAGE (110 min, 120 V, in 1 SDS buffer). 20. Detection of overexpressed T7Sav tetramers bound to B4F takes place under UV light (Gel Doc imager, Bio-Rad, Fig. 4a). 21. The gel is then gently shaken in protein-staining solution and afterward in protein-destaining solution. Both steps are performed for at least 2 h at RT. 22. Coomassie brilliant blue R-stained proteins are finally detected under white light (Fig. 4b).

3.5 Protein Purification Using the A¨KTAprime plus System

Equipment for affinity chromatography is composed of a XK ¨ KTAprime plus, both supplied 50/60 column and the instrument A by GE Healthcare. The column is filled with 250 mL (1 column volume, CV) of the 2-iminobiotin sepharose resin, which was made by following the protocol described by Wilchek and coworkers [50]. Due to its high binding capacity (25 mg/1 mL of resin), the affinity matrix can be used to purify several grams of T7Sav at once (see Note 20). Protein purification is monitored by UV absorbance at λ ¼ 280 nm. Depending on the amount of soluble streptavidin, the CFE is usually purified by one or two runs of affinity chromatography (see Note 21). 1. The affinity matrix is first washed with 1 CV of Milli-Q water. 2. The column is then equilibrated with 2 CVs of IBB buffer. Following this step, the CFE is loaded (see Note 22).

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3. Unbound protein (Flow through, FT) is collected and analyzed by SDS-PAGE. 4. The matrix is washed with at least 6 CVs of IBB buffer or until the UV detection signal is back to zero. 5. To elute T7Sav from the resin, 4 CVs of 1% (v/v) acetic acid are loaded (see Note 23). 6. As soon as the UV absorbance signal is back to zero, the resin is washed with 1 CV of Milli-Q water, followed by 1 CV of 20% (v/v) EtOH, for long-term storage of the matrix. 7. To remove acetic acid from the purified protein solution, the eluate is again subjected to dialysis. The dialysis bags are incubated first in 10 mM Tris–HCl dialysis buffer, followed by incubation in 15 L of DI-H2O, and finally by three more dialysis cycles in 15 L Milli-Q water. 8. The protein solution is finally subjected to high-speed centrifugation (20,000  g, 10 min) in order to remove precipitates and then freeze-dried using a lyophilizer (see Note 24). 3.6 Determination of FBS in Over-Expressed, Purified, and Lyophilized T7Sav

1. A black-walled 96-well microtiter plate (e.g., FluoroNunc, Thermo Scientific) is used for the measurement. Each T7Sav variant is measured in triplicate. Therefore, three protein samples are weighed and diluted to 2 μM using 0.1 M phosphate buffer with BSA. 2. Prior to use, the B4F stock solution is diluted to 40 μM. 3. For each protein sample, the following components displayed in the pipetting scheme (Table 1) are added to each well per row. 4. The solutions are thoroughly mixed. In order to remove air bubbles, the plate is subjected to centrifugation (1000  g, 2 min).

Table 1 Pipetting scheme for the measurement of free binding sites in streptavidin Amount (μL) Buffer/solution 40 μM B4F solution 0.1 M phosphate buffer with BSA

1

2

3

4

5

6

7

8

9

10

11

12

8

10

12

14

16

18

20

22

24

26

28

30

22

20

18

16

14

12

10

8

6

4

2

0

2 μM protein solution

100 100 100 100 100 100 100 100 100 100 100 100

Total

130 130 130 130 130 130 130 130 130 130 130 130

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5. The read-out (Fig. 5) takes place in a microplate reader. In the plate reader, the plate is shaken first for 30 s at 300 rpm prior to the determination of fluorescence (excitation wavelength: 485 nm, emission wavelength: 520 nm).

4

Notes 1. Protein expression using the ZYP-5052 medium is initiated by the inducer molecule lactose upon the depletion of glucose. However, the precise adjustment of the concentration of the two additional carbon sources, glycerol and glucose, is crucial to ensure maximum yields of T7Sav [38]. 2. Sav is a toxic protein for E. coli, since it leads to deprivation of the essential vitamin biotin during overexpression [48]. The OD600nm value after 24 h of cell growth is a first indicator whether protein expression was successful or not. OD600 nm values of 10–15 are desirable, since they guarantee a good compromise between cell growth and sufficient protein production. For small-scale production of T7Sav, the autoinducing medium has been successfully used as well [49]. 3. In order to avoid thermal degradation of the antibiotic, ampicillin is not immediately added to the autoclaved medium. The right temperature is reached when the bottle is still hot but can be touched with bare hands for a few seconds. 4. The sterilized stock solutions can also be kept for up to 6 months at RT. Prior to use and to avoid contamination of the medium, it is highly recommended to visually confirm if the solution is still clear. 5. Once made, the 10% (v/v) antifoam solution is stored at RT and used for several fermentations. Prior to each fermentation, the solution is autoclaved (20 min, 121  C, 1.5 bar) and chilled again. 6. The standard recipe for LBSOB includes glucose. In our preparations, glucose is not added. 7. Protease inhibitors, such as the hazardous compound phenylmethylsulfonylfluorid (PMSF), are not added to the lysis buffer. The E. coli strain BL21(DE3) that is often used for recombinant protein expression is deficient of the proteases OmpT and Lon, which are known to degrade foreign proteins [36, 51]. Protease-mediated degradation of T7Sav is barely observed neither by SDS-PAGE nor by ESI-MS at the very end of the entire procedure. 8. GdmCl is rather expensive and is used in very large quantities for our purposes. A dialysis tank with 15 L of 6 M GdmCl is reused for approximately 20 overexpressed proteins. Prior to each usage, the pH is readjusted to 1.5.

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9. The 20 mM Tris dialysis buffer, pH 7.4 and the IBB dialysis buffer, pH 10.8, which are used to renature T7Sav and to equilibrate the cell solution, respectively, are always freshly prepared after each usage in order to prevent contamination and microbial growth. To adjust the pH in a 15 L dialysis tank, a portable pH meter is used. 10. Originally, the IBB (dialysis) buffer was set to pH 9.8 [48]. This value was in some cases not high enough to ensure binding to immobilized 2-iminobiotin sepharose and was then increased to 10.8. 11. The N-terminal residue methionine is not considered for the calculation of the tetrameric weight of Sav, since this residue is cleaved by E. coli. 12. The purity of the B4F powder is claimed to be 95%. The concentration of the prepared B4F solution (εB4F ¼ 68,000/ M cm) is verified by ultraviolet-visible spectroscopy (UV-Vis) at a wavelength λB4F ¼ 495 nm. B4F is a light-sensitive compound. The aliquots are thus protected against light with aluminum foil. 13. When making competent cells, which do not grow in the presence of a selection marker, i.e., antibiotics, it is highly recommended to conduct all steps with highest care and under sterile conditions in order to avoid contamination. The efficiency of cell competence is strongly dependent on the temperature, which should not exceed 4  C. 14. Cell growth needs to be stopped early during the exponential growth phase in order to get highly competent cells. It is therefore crucial to not exceed the respective OD600nm value of 0.35, which is usually reached after 2.5 h at 37  C. 15. Incubation of cells in LB/LBSOB medium after the transformation of ampicillin-containing plasmids is usually allowed for 20–30 min. Properly sealed agar plates with colonies can be used for up to 4 weeks for protein production. 16. Protein overexpression was in some cases not observed, when glycerol stocks were used. Therefore, glycerol stocks are only made and stored as a plasmid backup but not for protein overexpression. Using freshly transformed E. coli cells results in a much more reliable production of T7Sav. 17. Successful cell lysis is reached when the CE has become a homogeneous solution that is free of clumps and not viscous. In contrast to a cell suspension, the CE has a slightly brown color and a strong smell. 18. Incubation in GdmCl, pH 1.5 is a very convenient step during Sav purification. While renaturation of T7Sav, particularly of our most commonly used Sav variants (WT, S112X and K121X

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mutants), is easily achieved by subsequent dialysis against Tris– HCl dialysis buffer, incubation in GdmCl leads to irreversible denaturation of most proteins other than Sav. The resulting CFE can be filtered more easily before it is applied to affinity chromatography. 19. The OD600nm value of harvested cells is always adjusted to 15 for cell lysis and the preparation for SDS-PAGE. This routine enables an easy comparison between different fermentor batches, i.e., different overexpressed T7Sav variants, in terms of quantity and functionality. 20. The binding capacity of the 2-iminobiotin sepharose resin is determined by a B4F titration assay. Binding of B4F to Sav results in fluorescence quenching. Upon saturation of all 4 binding sites of Sav with B4F, further addition of the B4F fluorophore leads to a sudden increase of fluorescence intensity (¼ turning point). At the turning point, the concentration of BF4 is equimolar to the concentration of monomeric Sav. In detail, 5 mL of a T7Sav WT protein solution (6.5 mg/mL) is loaded onto 1 mL of settled resin and the resulting flowthrough is applied another two times. The input solution, the last flow-through, and the elution are subjected to the B4F titration assay. The result of the B4F titration assay for the elution reveals the capacity of the column. The result is crosschecked for plausibility by subtracting the determined Sav in the flow-through from the determined Sav in the input. 21. Referring to the result of the SDS-PAGE gel (signal of soluble and functional T7Sav), the protein yield can be roughly estimated. The CFE can then be purified in one or several runs of affinity chromatography depending on protein yield and column capacity (see also Note 23). 22. In cases of low protein yield, up to 1.5 L of CFE has to be loaded onto our 2-iminobiotin sepharose column. To load ¨ KTAprime such a large volume via the injection valve of the A Plus is extremely cumbersome. With this instrument it is however possible to use its buffer valve and system pump to load the CFE. In order to keep the system pump in a good condition and clean, it is mandatory to load only filtered (0.22 μm pore-sized filter) and freshly prepared CFE. 23. If the affinity matrix is saturated, it can happen that protein precipitates in the collection tubes during elution. In order to prevent precipitation upon elution, it might be advisable to keep the protein loading below the capacity of the resin, e.g., by 50%. 24. The protein powder can be kept for many years at 4  C without any loss of binding affinity or catalytic performance of prepared artificial metalloenzymes.

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25. For some T7Sav mutants the B4F assay cannot be used to determine the number of FBS. Although these mutants bind 2-iminobiotin during affinity chromatography or B4F during SDS-PAGE, we do not observe an accompanying decrease in fluorescence intensity [39]. In these cases, another spectrophotometric method can be applied [52–54]. The assay is based on the displacement of the organic dye 2-hydroxyazobenzen-40 -carboxylic acid (HABA) from Sav upon the addition of biotin, which has a stronger affinity to the protein. The HABA-Sav complex absorbs light at a wavelength of λ ¼ 506 nm. The addition of biotin leads to replacement of HABA in the biotin binding site and results in decreased absorbance. References 1. Schwizer F, Okamoto Y, Heinisch T, Gu Y, Pellizzoni MM, Lebrun V, Reuter R, Ko¨hler V, Lewis JC, Ward TR (2018) Artificial metalloenzymes: reaction scope and optimization strategies. Chem Rev 118(1):142–231. https://doi.org/10.1021/acs.chemrev. 7b00014 2. Ward TR (2011) Artificial metalloenzymes based on the biotin–avidin technology: enantioselective catalysis and beyond. Acc Chem Res 44(1):47–57. https://doi.org/10.1021/ ar100099u 3. Liang AD, Serrano-Plana J, Peterson RL, Ward TR (2019) Artificial metalloenzymes based on the biotin–streptavidin technology: enzymatic cascades and directed evolution. Acc Chem Res 52(3):585–595. https://doi.org/10.1021/ acs.accounts.8b00618 4. Heinisch T, Ward TR (2016) Artificial metalloenzymes based on the biotin–streptavidin technology: challenges and opportunities. Acc Chem Res 49(9):1711–1721. https://doi. org/10.1021/acs.accounts.6b00235 5. Kohler V, Wilson YM, Durrenberger M, Ghislieri D, Churakova E, Quinto T, Knorr L, Haussinger D, Hollmann F, Turner NJ (2013) Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes. Nat Chem 5(2):93–99 6. M€a€att€a JAE, Eisenberg-Domovich Y, Nordlund HR, Hayouka R, Kulomaa MS, Livnah O, Hyto¨nen VP (2011) Chimeric avidin shows stability against harsh chemical conditions—biochemical analysis and 3D structure. Biotechnol Bioeng 108 (3):481–490. https://doi.org/10.1002/bit. 22962 7. Holmberg A, Blomstergren A, Nord O, Lukacs M, Lundeberg J, Uhlen M (2005) The biotin-streptavidin interaction can be reversibly broken using water at elevated

temperatures. Electrophoresis 26 (3):501–510. https://doi.org/10.1002/elps. 200410070 8. Sano T, Cantor CR (1990) Expression of a cloned streptavidin gene in Escherichia coli. Proc Natl Acad Sci U S A 87(1):142–146. https://doi.org/10.1073/pnas.87.1.142 9. Weber P, Ohlendorf D, Wendoloski J, Salemme F (1989) Structural origins of highaffinity biotin binding to streptavidin. Science 243(4887):85–88. https://doi.org/10.1126/ science.2911722 10. Dundas CM, Demonte D, Park S (2013) Streptavidin-biotin technology: improvements and innovations in chemical and biological applications. Appl Microbiol Biotechnol 97 (21):9343–9353. https://doi.org/10.1007/ s00253-013-5232-z 11. Wilchek M, Bayer EA (1988) The avidin-biotin complex in bioanalytical applications. Anal Biochem 171(1):1–32. https://doi.org/10. 1016/0003-2697(88)90120-0 12. Wilson YM, Durrenberger M, Nogueira ES, Ward TR (2014) Neutralizing the detrimental effect of glutathione on precious metal catalysts. J Am Chem Soc 136(25):8928–8932. https://doi.org/10.1021/ja500613n 13. Zhao J, Rebelein JG, Mallin H, Trindler C, Pellizzoni MM, Ward TR (2018) Genetic engineering of an artificial metalloenzyme for transfer hydrogenation of a self-immolative substrate in Escherichia coli’s periplasm. J Am Chem Soc 140(41):13171–13175. https:// doi.org/10.1021/jacs.8b07189 14. Creus M, Pordea A, Rossel T, Sardo A, Letondor C, Ivanova A, LeTrong I, Stenkamp RE, Ward TR (2008) X-ray structure and designed evolution of an artificial transfer hydrogenase. Angew Chem Int Ed 47

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activation. Science 338(6106):500–503. https://doi.org/10.1126/science.1226132 24. Chambers JM, Lindqvist LM, Webb A, Huang DCS, Savage GP, Rizzacasa MA (2013) Synthesis of biotinylated episilvestrol: highly selective targeting of the translation factors eIF4AI/II. Org Lett 15(6):1406–1409. https://doi.org/10.1021/ol400401d 25. Zimbron JM, Heinisch T, Schmid M, Hamels D, Nogueira ES, Schirmer T, Ward TR (2013) A dual anchoring strategy for the localization and activation of artificial metalloenzymes based on the biotin-streptavidin technology. J Am Chem Soc 135 (14):5384–5388. https://doi.org/10.1021/ ja309974s 26. Wilson ME, Whitesides GM (1978) Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site specific modification with a diphosphinerhodium(I) moiety. J Am Chem Soc 100:306–307 27. Knowles WS (2002) Asymmetric hydrogenations (nobel lecture). Angew Chem Int Ed 41 (12):1998–2007. https://doi.org/10.1002/ 1521-3773(20020617)41:123.0.CO;2-8 28. Noyori R, Hashiguchi S (1997) Asymmetric transfer hydrogenation catalyzed by chiral ruthenium complexes. Acc Chem Res 30 (2):97–102 29. Mashima K, Abe T, Tani K (1998) The halfsandwich hydride and 16-electron complexes of rhodium and iridium containing (1S,2S)N-(p-toluenesulfonyl)-1,2-diphenylethylenediamine: relevant to asymmetric transfer hydrogenation. Chem Lett 12:1201–1202. https:// doi.org/10.1246/cl.1998.1201 30. Garber SB, Kingsbury JS, Gray BL, Hoveyda AH (2000) Efficient and recyclable monomeric and dendritic Ru-based metathesis catalysts. J Am Chem Soc 122(34):8168–8179. https:// doi.org/10.1021/ja001179g 31. Gessler S, Randl S, Blechert S (2000) Synthesis and metathesis reactions of a phosphine-free dihydroimidazole carbene ruthenium complex. Tetrahedron Lett 41(51):9973–9976. https:// doi.org/10.1016/S0040-4039(00)01808-6 32. Lo C, Ringenberg MR, Gnandt D, Wilson Y, Ward TR (2011) Artificial metalloenzymes for olefin metathesis based on the biotin-(strept) avidin technology. Chem Commun (Cambridge, U K) 47(44):12065–12067. https:// doi.org/10.1039/c1cc15004a 33. Quinto T, Schwizer F, Zimbron JM, Morina A, Koehler V, Ward TR (2014) Expanding the chemical diversity in artificial imine reductases based on the biotin-streptavidin technology. ChemCatChem 6(4):1010–1014. https:// doi.org/10.1002/cctc.201300825

Streptavidin-Based Artificial Metalloenzymes 34. Okamoto Y, Ko¨hler V, Ward TR (2016) An NAD(P)H-dependent artificial transfer hydrogenase for multienzymatic cascades. J Am Chem Soc 138(18):5781–5784. https:// doi.org/10.1021/jacs.6b02470 35. Chaiet L, Miller TW, Tausig F, Wolf FJ (1963) Antibiotic MSD-235. II. Separation and purification of synergistic components. Antimicrob Agents Chemother (Bethesda) 161:28–32 36. Joseph BC, Pichaimuthu S, Srimeenakshi S, Murthy M, Selvakumar K, Ganesan M, Manjunath SR (2015) An overview of the parameters for recombinant protein expression in Escherichia coli. J Cell Sci Ther 6(5):1000221/ 1000221–1000221/1000227. https://doi. org/10.4172/2157-7013.1000221 37. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189(1):113–130. https://doi.org/10. 1016/0022-2836(86)90385-2 38. Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41(1):207–234. https://doi.org/10.1016/j.pep.2005.01.016 39. Humbert N, Zocchi A, Ward TR (2005) Electrophoretic behavior of streptavidin complexed to a biotinylated probe: a functional screening assay for biotin-binding proteins. Electrophoresis 26(1):47–52. https://doi.org/10.1002/ elps.200406148 40. Sano T, Pandori MW, Chen X, Smith CL, Cantor CR (1995) Recombinant core streptavidins. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules. J Biol Chem 270(47):28204–28209 41. Le Trong I, Humbert N, Ward TR, Stenkamp RE (2006) Crystallographic analysis of a fulllength streptavidin with its C-terminal polypeptide bound in the biotin binding site. J Mol Biol 356(3):738–745. https://doi.org/ 10.1016/j.jmb.2005.11.086 42. Gallizia A, De Lalla C, Nardone E, Santambrogio P, Brandazza A, Sidoli A, Arosio P (1998) Production of a soluble and functional recombinant streptavidin in Escherichia coli. Protein Expr Purif 14(2):192–196. https://doi.org/10.1006/prep.1998.0930 43. Sano T, Cantor CR (1995) Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin. Proc Natl Acad Sci U S A 92

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Chapter 13 Integrated Bacterial Production and Functional Screening of Expanded Cyclic Peptide Libraries for Identifying Chemical Rescuers of Pathogenic Protein Misfolding and Aggregation Dafni C. Delivoria and Georgios Skretas Abstract Protein misfolding and aggregation are defining features of a wide range of human conditions, collectively termed protein misfolding diseases. These include disorders with diverse pathologies and symptoms, such as Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes, the vast majority of which impose a very high socio-economic burden on humanity and remain incurable to date. To address this unmet medical need, we report here a simple and high-throughput system for identifying macrocyclic rescuers of protein misfolding. In this system, Escherichia coli cells are genetically engineered in order to perform two simultaneous tasks: (1) produce combinatorial libraries of head-to-tail cyclic oligopeptides using proteinsplicing technology and (2) enable the identification of the bioactive cyclic peptides that correct the problematic folding and/or inhibit the aggregation of disease-associated misfolding-prone proteins using a genetic assay that links the folding of the target protein to a fluorescent phenotype. In this way, the bioactive cyclic peptide hits are identified in an ultrahigh-throughput manner using flow cytometric cell sorting, thus significantly decreasing the overall cost, time, and complexity of early drug discovery for these notorious diseases. Key words Protein misfolding diseases, Protein aggregation, High-throughput screening, SICLOPPS technology, Cyclic peptides, Directed evolution

1

Introduction In the past decades, protein misfolding and aggregation have been associated with numerous human diseases of diverse pathologies that have been collectively termed protein misfolding diseases (PMDs), proteinopathies or conformational diseases [1–4]. These can be (1) familial, such as Huntington’s disease (HD), (2) sporadic, such as most cases of Parkinson’s disease (PD), (3) iatrogenic, such as dialysis-related amyloidosis, and (4) infectious, such as Creutzfeldt-Jacob disease [1]. All PMDs, however, and irrespective

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_13, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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of their nature, have been associated with the aberrant folding and/or aggregation of one or more misfolded proteins (MisPs) [1]. Importantly, although for some PMDs symptomatic therapies do exist, there are no approved disease-modifying therapies that can prevent, delay, or reverse the progression of the disease for the vast majority of these conditions [5–7]. Recently, there has been a revival of interest in peptide-based molecules in drug discovery and development, with an emphasis in macrocyclic peptides. These exhibit the favorable characteristic of linear peptides, such as high specificity and low in vivo toxicity but, at the same time, usually do not share their limitations, such as high protease susceptibility and low membrane permeability [8]. Combinatorial libraries of macrocyclic peptides can be produced either by synthetic methods, including DNA-encoded library technologies [9, 10], or by genetic methods, including (1) display technologies, such as phage [11, 12], yeast [13], bacteria [14], messenger RNA [15–18], and ribosome display [19], and (2) an intein-based approach termed “split intein-mediated circular ligation of peptides and proteins” (SICLOPPS) that enables the production of head-totail cyclic peptides of small ring sizes inside living cells [20, 21]. Inteins are protein splicing elements that are widely used in protein engineering and biotechnology, in applications such as tag-less protein purification, segmental isotopic labelling of large proteins for nuclear magnetic resonance (NMR) studies, in vitro and in vivo protein semi-synthesis, development of molecular switches using conditional protein splicing, and, last but not least, head-to-tail cyclization of peptides and proteins [22, 23]. Protein splicing is an autocatalytic process in which an intervening polypeptide (intein) is self-excised, while concomitantly ligating its two flanking sequences (exteins) with a native amide bond (Fig. 1a). While most inteins are encoded by one gene forming a contiguous domain, some inteins are naturally, or engineered to be, encoded by two separate genes forming a split intein, which comprises an N-terminal (IN) and a C-terminal domain (IC) [24]. These domains remain inactive until encountering their counterpart, whereupon they undergo protein trans-splicing (Fig. 1b). The SICLOPPS technique utilizes a rearranged split intein so that the IC precedes the IN flanking a peptide sequence in the form IC-peptide-IN [25]. Upon the interaction of the two intein domains, the two ends of the intervening peptide are ligated and a head-to-tail cyclic peptide is released (Fig. 1c). The SICLOPPS technology has been successfully employed up to now in E. coli, yeasts, human cells, and Caenorhabditis. elegans; and combinatorial libraries with up to 108 members for E. coli [26], 106 for yeasts [27], and 104 for human cells [28] have been constructed and subsequently screened against different targets. However, compared to other competitive techniques, such as mRNA display, the diversity limits of the cyclic peptide libraries produced

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Fig. 1 Schematic of protein splicing in (a) cis, (b) trans, and (c) using the SICLOPPS technology

via SICLOPPS are significantly smaller, restricted by the number of possible combinations of amino acids allowed by the specific library design and the maximum transformation efficiency of the host organism (~109–1010 transformants for E. coli, ~106 transformants for Saccharomyces cerevisiae, ~104 transformants for mammalian cells). Nevertheless, this method is highly valuable as the cyclic peptide libraries are produced inside cells and can therefore be screened using cell-based functional assays [25, 29]. This is an important advantage as cyclic peptides produced by this approach can be selected according to their bioactivity and not just their binding affinity to a given target, eliminating the possibility of identifying cyclic peptides that are strong binders but are either completely inactive or with the opposite effect than originally intended. Microbial hosts, such as bacteria and yeasts, offer the opportunity of studying protein aggregation in simplified, but still physiologically relevant conditions, as several protein folding and misfolding features can be reliably generated in both prokaryotic and eukaryotic microorganisms. Most importantly, microbial hosts enable the development of high-throughput and affordable screens, which result in biologically active compound hits. Indeed,

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Fig. 2 Schematic of the MisP-GFP genetic system for monitoring MisP folding and aggregation. MisP diseaseassociated misfolding-prone protein, GFP green fluorescent protein

a wide range of microbial screens have been developed for monitoring and rescuing protein misfolding, thoroughly reviewed elsewhere [30]. A notable example is the development of a yeast model of PD, where the increased expression of α-synuclein resulted in the formation of inclusion bodies and severe cytotoxicity [31]. This system was later used to screen various small molecule libraries [31– 33], as well as a cyclic octapeptide library produced via the aforementioned SICLOPPS technology [27], resulting in the identification of several biologically active compounds against PD. Monitoring of protein folding and aggregation can be performed using E. coli cells by coupling a disease-associated MisP with a reporter protein, such as the green fluorescent protein (GFP), in such a way that its function will depend on the correct folding of MisP. Due to the aggregation propensity of MisP, overexpression of MisP-GFP in E. coli results in the accumulation of insoluble inclusion bodies exhibiting decreased fluorescence (Fig. 2). Contrarily, conditions that inhibit protein misfolding and aggregation result in the production of soluble MisP-GFP fusions that exhibit increased levels of fluorescence. This method was first

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utilized by Hecht and co-workers, who coupled the Alzheimer’s disease–associated amyloid-β peptide (Aβ42) to GFP and showed that bacterial overexpression of Aβ42-GFP resulted in intracellular insoluble aggregates and lack of green fluorescence [34]. Subsequently, this assay has been utilized by several groups to screen small-molecule or combinatorial libraries for the identification of Aβ42 aggregation inhibitors [35–37]. Interestingly, the GFP-based screen has been utilized to monitor the aggregation of other disease-associated MisPs, such as the islet amyloid polypeptide (IAPP) [38] and mutants of p53 [39], which are associated with type 2 diabetes and cancer, respectively. Toward accelerating the early drug discovery process and addressing the imperative medical need for effective therapeutics against PMDs, we have reported the development of a novel ultrahigh-throughput synthetic biology platform, where E. coli cells are engineered in order to perform two simultaneous tasks: (1) produce very large combinatorial libraries of head-to-tail cyclic peptides using the SICLOPPS technology and (2) enable their direct functional screening by employing the MisP-GFP genetic assay coupled with flow cytometric sorting [40]. This approach enables the identification of cyclic peptides with the ability to bind the MisP of interest and rescue its misfolding and/or aggregation, by monitoring their effect on the fluorescence of the recombinant MisP-GFP fusions. Specifically, since the bacterial fluorescence of E. coli cells producing MisP-GFP fusions is strongly correlated to the aggregation propensity of the target MisP, cells producing bioactive cyclic peptides will exhibit significantly increased levels of GFP fluorescence. As both cyclic peptide production and their functional screening are performed inside living cells, bacterial clones producing such cyclic peptides can be isolated in an ultrahigh-throughput manner using fluorescence-activated cell sorting (FACS), therefore enabling the rapid and facile identification of putative rescuers of protein misfolding (Fig. 3) [40]. We have successfully utilized this platform to produce combinatorial libraries of random head-to-tail cyclic tetra-, penta-, hexa-, and heptapeptides with a total diversity of over 200 million different molecules and screen them against the misfolding and aggregation of Aβ42 and mutant Cu/Zn superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis [26, 40]. While this diversity already exceeds the potential of other direct functional screens reported to date, the high transformation efficiency of E. coli cells enables the construction of even larger libraries, consisting of up to 1010 macrocyclic peptides with both natural and artificial amino acids, thus significantly increasing the possibility of identifying bioactive hits against these devastating diseases. Finally, but very importantly, this bacterial system can be employed against different target diseases as it takes advantage of their common molecular origin, i.e., protein misfolding and

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Fig. 3 Schematic of the utilized bacterial platform for discovering inhibitors of protein aggregation. pMisP-GFP plasmid encoding a misfolded protein-GFP fusion, pSICLOPPS-NuX1X2-Xn vector library encoding the combinatorial cyclo-NuX1X2-Xn, Nu Cys, Ser, or Thr, X any of the 20 natural amino acids, n any number equal to or greater than 3 (see Note 48), FSC-H forward scatter, SSC-H side scatter, P sorting gate

aggregation. Indeed, besides Aβ42 and SOD1, we have already identified putative rescuers of the misfolding of mutated variants of p53 and huntingtin, associated with cancer and Huntington’s disease, respectively (unpublished data). Overall, the approach presented herein represents a highly versatile ultrahigh-throughput strategy that enables the facile and cost-effective investigation of libraries with greatly expanded diversities and the discovery and characterization of potent rescuers of protein misfolding and/or aggregation, associated with a broad range of PMDs.

2

Materials

2.1 Growth Media, Antibiotics, Inducers of Protein Production, and Common Buffers

1. Luria-Bertani broth (LB): 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl. 2. Super optimal broth (SOB): 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 2.5 mM KCl, 20 mM MgSO4. 3. 2
YT medium: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0. 4. Antibiotics depending on the resistance of the respective plasmid (stock solutions of 50 mg/mL kanamycin (Kan), 34 mg/ mL chloramphenicol (Cm), 100 mg/mL ampicilin (Amp), etc.). 5. Inducers of protein production depending on the promoter of the respective plasmid (stock solutions of 20% w/v L (+)arabinose, 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG), 20 mg/L anhydrotetracycline (aTc), etc.).

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6. 1
Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer: 2 M Tris base, 1 M glacial acetic acid, 50 nM EDTA, pH 8.0. 7. Sodium dodecyl sulfate (SDS) sample buffer (6
): 6% w/v SDS, 300 mM Tris–HCl, 15% v/v glycerol, 0.01% w/v bromophenol blue, 10% v/v β-mercaptoethanol, pH 6.8. 8. Tris-buffered saline with Tween-20 (TBST): 20 mM Tris base, 150 mM NaCl, 0.1% v/v Tween-20, pH 7.4. 9. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. 10. Flow cytometer sheath fluid. 2.2

E. coli Strains

1. Electrocompetent E. coli cells for the production of the pSICLOPPS library, prepared in-house or purchased (e.g., NEB 10-beta from New England Biolabs) (see Note 1). 2. E. coli cells for the expression of MisP-GFP (e.g., BL21(DE3), Tuner(DE3) and Origami2(DE3)) (see Note 2).

2.3

Plasmid Vectors

1. pARCBD-p vector derived by Scott et al. [20], containing the Synechocystis sp PCC6803 DnaE (Ssp DnaE) split intein fused with a chitin-binding domain (CBD) (see Note 3). 2. Appropriate vector expressing the MisP-GFP construct under a (strong) promoter (see Notes 4–6). 3. pET-28a (+) expression vector.

2.4 Enzymes Used for DNA Cloning

1. High-fidelity DNA polymerase and its respective reaction buffer (e.g., Q5 High-fidelity DNA polymerase). 2. Standard Taq DNA polymerase and its respective reaction buffer. 3. NcoI, BglI, BsrGI, and HindIII restriction enzymes and their suitable reaction buffers. 4. Thermosensitive alkaline phosphatase (e.g., Shrimp alkaline phosphatase from NEB (rSAP) or FastAp from Thermo Scientific). 5. T4 DNA ligase and T4 DNA ligase buffer.

2.5 Primers Used for DNA Cloning and Sequencing

1. Forward KanR primer: AAAAAAGCCAATGGGGCATGAGC CATATTCAACGGGAAAC. 2. Reverse KanR primer: TTTTTTAAGCTTTTAGAAAAACT CATCGAGC. 3. Degenerate forward PCR primer for the construction of the cyclo-CysX1X2-Xn sub-library: GGAATTCGCCAATGGG GCGATCGCCCACAATTGC(NNS)nTGCTTAAGTTTTGGC (see Note 7).

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4. Degenerate forward PCR primer for the construction of the cyclo-SerX1X2-Xn sub-library: GGAATTCGCCAATGGGG CGATCGCCCACAATAGC(NNS)nTGCTTAAGTTTTGGC (see Note 7). 5. Degenerate forward PCR primer for the construction of the cyclo-ThrX1X2-Xn sub-library: GGAATTCGCCAATGGG GCGATCGCCCACAATACC(NNS)nTGCTTAAGTTTTGGC (see Note 7). 6. Reverse CBD primer: GCTGCCACAAGG.

AAAAAAAAGCTTTCATTGAA

7. Forward Cys zipper primer: AAAAAAGCCAATGGGGCGA TCGCCCACAATTGC (see Note 8). 8. Forward Ser zipper primer: AAAAAAGCCAATGGGGCGAT CGCCCACAATAGC (see Note 8). 9. Forward Thr zipper primer: AAAAAAGCCAATGGGGCGA TCGCCCACAATACC (see Note 8). 10. pARCBDseqFor: CTATAACTATGGCTGGAATG. 11. p15AoriSeqRev: GCCCCATACGATATAAGTTG. 12. SspDnaE(H24L/F26A)rev: TTTTTTGCCCCATTGGCTAG CAGAGCATTAAGGTCTTGGGGAAGACCAATAT. 2.6 SDS-PAGE/ Western Blot Reagents

1. 4–20% precast polyacrylamide gel. 2. Polyvinylidene fluoride (PVDF) membrane. 3. Non-fat dry milk. 4. Mouse anti-CBD antibody (e.g., from NEB, USA) at 1:100,000 dilution. 5. Mouse anti-GFP antibody (e.g., from Clontech, USA) at 1:20,000 dilution. 6. Horseradish peroxidase (HRP)–conjugated goat anti-mouse secondary antibody (e.g., from BioRad, USA) at 1:4000 dilution.

2.7 Additional Reagents

1. Deoxynucleotide (dNTP) mix consisting of 10 mM of dATP, dTTP, dGTP, and dCTP. 2. Plasmid Mini and Midi kits (e.g., from Qiagen or Macherey Nagel). 3. PCR and gel clean-up kits (e.g., from Qiagen or Macherey Nagel). 4. Agarose powder, preferably of low electroendoosmosis (EEO). 5. Dye for nucleic acid straining (e.g., ethidium bromide (EtBr) or SYGR Green) (see Note 9). 6. DNA gel loading dye.

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7. PCR-free Illumina-compatible library preparation kit (e.g., ClaSeek kit from Thermo Scientific). 8. Quantitative PCR (qPCR) next-generation sequencing (NGS) library quantification kit (e.g., from Agilent technologies). 9. High-sensitivity DNA kit (e.g., from Agilent Technologies). 10. Black, flat-bottom 96-well plate (e.g., FLUOTRAC 200 from Greiner). 2.8

Software

1. FlowJo v10 and FACSDiva v8.0.1. 2. Sequencing read aligner tool (e.g., Bowtie v.2.2.8 [41]). 3. Clustering tool for large databases of nucleotide sequences (e.g., CD-HIT [42, 43]). 4. Clustering tool using the fully interconnected clusters (cliques) method (e.g., Immune Epitope Database (IEDB) clustering tool (http://tools.iedb.org/cluster/) [44]). 5. Network visualization software (e.g., Gephi multiplatform [45]).

2.9

Equipment

1. Thermal cycler. 2. UV spectrometer. 3. Horizontal electrophoresis electrophoresis.

apparatus

for

DNA

gel

4. Vertical electrophoresis apparatus for protein electrophoresis. 5. Protein transfer system. 6. Imaging system for DNA and protein analysis (e.g. ChemiDocIt2 from UVP). 7. High-speed refrigerated microcentrifuge for 1.5 mL tubes. 8. Incubator shaker. 9. Water bath sonicator (e.g., Bioruptor sonication system from Diagenode). 10. Flow cytometer, equipped with a 488 nm solid state laser for excitation and a 530/30 nm band pass filter for detection (e.g., BD FACSAria III system). 11. 96-well plate reader (e.g., TECAN Safire II-Basic). 12. High-throughput sequencing platform (e.g., Ion Torrent).

3

Methods Several protocol can be found in the literature for the production of cyclic peptide libraries using the SICLOPPS technology [46– 48]. Taking these into consideration and after making certain modifications, we have resulted in the following procedure:

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3.1 Construction of Libraries of Head-to-Tail Cyclic Peptides in E. coli

1. Construct the auxiliary pSICLOPPSKan vector This procedure is optional but highly recommended as using an auxiliary vector, such as the pSICLOPPSKan vector, for the construction of the SICLOPPS library enables the facile estimation of the cloning success rate (see Note 10). (a) Prepare and run a PCR reaction according to the manufacturer’s instructions using the pET-28a(+) vector as a template and forward and reverse KanR primers. Purify the PCR product using a PCR purification kit. (b) Digest the purified PCR product and the pARCBD-pvector with BglI and HindIII according to the manufacturer’s instructions. (c) Construct the pSICLOPPSKan vector by ligating the KanR gene and the pARCBD-p vector using a T4 DNA ligase and according to the manufacturer’s instructions. (d) Transform E. coli cells with the newly constructed pSICLOPPSKan and purify the plasmid using a plasmid purification kit. 2. Prepare a PCR reaction as described in Table 1 and perform the reaction using the thermocycling conditions in Table 2 (see Note 11). 3. Verify successful DNA amplification (~560 bp) by running a 1% agarose gel with 1
TAE buffer and purify the PCR products using a PCR purification kit. 4. Prepare and run a second PCR reaction as in step 2 with the only differences being the use of (1) a forward zipper primer and (2) 100 ng of the PCR product from step (3) as a template. Repeat step 3.

Table 1 PCR reagents for construction of the pSICLOPPS library Reagent

50 μL reaction

Final concentration

Q5 reaction buffer (5
)

10

1


Q5 high GC enhancer (5
)

10

1


dNTP mix (10 mM each)

1

200 μM

Degenerate Forward primer (10 μM)

2.5

0.5 μM

Reverse CBD primer (10 μM)

2.5

0.5 μM

Template DNA (pARCBD-p)

Variable

100 ng

Q5 High-fidelity DNA polymerase

0.5

1U

ddH2O

To 50 μL

–

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Table 2 PCR conditions for construction of the pSICLOPPS library construction Step

Temperature ( C)

Time

Initial denaturation

98

30 s

98 72 72

10 s 30 s 1 min

72

2 min

Cycles 1–30

Final extension Hold

Denaturation Annealing Extension

4

–

5. Digest the purified PCR products and the pSICLOPPSKan vector using the restriction enzymes BglI and HindIII in NEBuffer 3.1 for 5 h at 37  C (see Note 12). 6. Inactivate the restriction enzymes by heating at 80  C for 20 min (see Note 13). 7. Run the digested DNA on a 1% agarose gel with 1
TAE buffer, and purify the appropriate DNA bands using a DNA gel extraction kit. 8. Dephosphorylate the digested vector using alkaline phosphatase, such as FastAP from Thermo Scientific, according to manufacturer’s instructions (see Note 13). 9. Determine the concentration of the digested PCR products and digested/dephosphorylated pSICLOPPSKan vector by measuring their UV absorbance at 260 nm. 10. Prepare ligation reactions consisting of the digested PCR product, the digested and dephosphorylated pSICLOPPSKan vector, T4 DNA ligase, and 1
T4 DNA ligase buffer in double distilled water (ddH2O); and incubate at 16  C for 4 h (see Note 14). Always prepare a control reaction in which the insert fraction is substituted with ddH2O. 11. Inactivate the T4 DNA ligase by heating at 65  C for 20 min (see Note 13). 12. Perform buffer exchange/desalting of the ligation reactions using spin columns and elute in an appropriate volume of ddH2O (see Note 15). 13. Add 10 μL of the purified ligation reaction in 60 μL of electrocompetent E. coli cells, and transfer to a chilled electroporation cuvette on ice (see Notes 16 and 17). 14. Thoroughly wipe the electrodes of the cuvette to remove any residual moisture and ice (see Note 18). 15. Apply a short high-voltage electrical pulse (generally 1.8 kV for cuvettes with a 0.1 cm gap).

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16. Immediately add 1 mL of SOB to the cuvette, gently resuspend the cells, and transfer into a polypropylene culture tube (see Notes 19 and 20). 17. Recover cells at 37  C with shaking for 1 h. 18. Plate cells containing the constructed pSICLOPPS libraries or the control ligation reaction on pre-heated LB agar containing 34 μg/mL Cm (see Note 21). In each case, also plate serial dilutions that will enable the estimation of the transformation efficiency and the size of the constructed pSICLOPPS library. Incubate all plates at 37  C for 12–16 h. 19. Estimate the number of independent transformants for each library by counting the colonies on the serial dilution plates. From this number subtract the amount of independent transformants found by other means, e.g., colony PCR or diagnostic digestion, to contain reclosed vector (see below). The final number should be at least 3–10 times larger than the theoretical library diversity in order to achieve full library coverage. 20. Detach the cells from the agar by scrapping and wash each plate thoroughly with LB. 21. Collect and vortex the cell suspension in order to break any bacterial clumps. 22. Prepare a sub-culture of an optical density at 600 nm (OD600) equal to 0.1 and incubate overnight at 37  C (see Note 22). 23. Purify the pSICLOPPS library using a Midiprep plasmid kit (Fig. 4). 24. Store the isolated pSICLOPPS library at 20  C. 3.2 Quality Assessment of the Constructed pSICLOPPS Library

The quality of the constructed pSICLOPPS library can be assessed using: (1) molecular biology techniques, including colony PCR, diagnostic digestion, and SDS-PAGE/western blot analysis and (2) deep sequencing analysis. These techniques are complementary as: (1) colony PCR and diagnostic digestion offer the possibility of detecting the presence of the correct insert and estimating the cloning success rate, (2) SDS-PAGE/western blot analysis permit the detection of the overexpressed precursor protein and its subsequent processing to yield the cyclic peptide, and (3) deep sequencing analysis offers the possibility of evaluating the diversity of the constructed library and identifying biases in the peptide sequences in greater detail. 1. Transform E. coli cells with the constructed pSICLOPPS library, and plate on LB agar containing 34 μg/mL Cm. 2. Inoculate liquid LB cultures supplemented with Cm with randomly selected individual colonies, and incubate at 37 oC for 16 h.

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Fig. 4 Schematic of the generation of the cyclic-NuX1X2-Xn peptide libraries via the SICLOPPS technology. (Left) Representation of the pSICLOPPS-NuX1X2-Xn vector library encoding the combinatorial heptapeptide library cyclo-NuX1X2-Xn. Nu Cys, Ser, or Thr, X any of the 20 natural amino acids, n any number equal to or greater than 3, NNS randomized codons, where N¼A, T, C or G and S¼G or C, IC C-terminal domain of the Ssp DnaE split-intein, IN N-terminal domain of the Ssp DnaE split-intein, CBD chitin-binding domain. (Right) Peptide cyclization using the SICLOPPS construct. Upon interaction between the two intein domains IC and IN, the encoded IC-NuX1X2-Xn-IN-CBD fusions undergo intein splicing and peptide cyclization, leading to the production of the cyclo-NuX1X2-Xn library

Table 3 Reagents for colony PCR Reagent

20 μL reaction

Final concentration

Standard Taq reaction buffer (10
)

2

1


dNTP mix (10 mM each)

0.4

200 μM

pARCBDseqFor forward primer (10 μM)

0.4

0.2 μM

p15AoriseqRev reverse primer (10 μM)

0.4

0.2 μM

Template DNA (overnight liquid culture)

2

variable

Taq DNA polymerase

0.1

0.5 U

ddH2O

To 20 μL

–

3. For colony PCR experiments: (a) Prepare PCR reactions as described in Table 3 and using 2 μL of each overnight culture as a template (see Notes 23–25). (b) Run the PCR reactions using the protocol from Table 4.

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Table 4 Standard conditions for colony PCR Step

Temperature ( C)

Time

Initial denaturation

95

30 s

95 55 68

30 s 1 min 1.5 min

68

5 min

Cycles 1–30

Final extension

Denaturation Annealing Extension

Hold

4

–

(c) Perform DNA electrophoresis of 10 μL of the PCR reaction on a 1% agarose gel with 1
TAE buffer. When using pSICLOPPSKan as a starting vector, a positive result is indicated by a band of ~880 bp if backbone-specific primers are used, such as pARCBDseqFor and p15AoriseqRev, or ~560 bp in the case of insert-specific primers. 4. For diagnostic digestion: (a) Purify the plasmid DNA for the randomly selected clones using a plasmid isolation kit. (b) Digest 5 μL of the purified plasmid with the restriction enzymes BglI and HindIII, and incubate for 30 min at 37 oC (see Note 12). (c) Load the reactions on a 1% agarose gel after the addition of the appropriate loading dye, and perform DNA electrophoresis in 1
TAE buffer. A positive result is indicated by a band of ~560 bp, while a negative result (i.e., re-closed pSICLOPPS vector) by a band of ~820 bp, when using pSICLOPPSKan as a starting vector. 5. For SDS-PAGE/western blot analysis: (a) Prepare 1% subcultures in 5 mL LB supplemented with Cm, and incubate at 37 oC with shaking until an OD600 of 0.3–0.5. (b) Induce the production of the tetra-partite IC-peptide-INCBD fusion by adding 0.002% w/v L (+)-arabinose, and incubate at 37 oC with shaking for 3 h. (c) Harvest cells corresponding to 1 mL of each culture with OD600 ¼ 3 by centrifugation at 6000
g for 2 min. (d) Resuspend cells in 100 μL PBS and lyse by brief sonication using a water bath sonicator, such as the Bioruptor sonication system from Diagenode. (e) Centrifuge the total cell lysates (10,000
g, 30 min, 4 oC), and collect the soluble supernatant.

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(f) Add 20 μL of 6
Laemmli SDS sample buffer (1:6 dilution), and boil samples for 10 min at 95 oC. (g) Load 10 μL of each sample, and run on a 4–20% precast polyacrylamide gel. (h) Transfer the proteins from the polyacrylamide gel onto a PVDF membrane. (i) Block the membrane with 5% non-fat dry milk in TBST for 1 h at room temperature. (j) Wash the membrane at least three times for 10 min with fresh TBST (see Note 26). (k) Incubate the membrane with a mouse anti-CBD primary antibody in 1:100,000 dilution in TBST containing 0.5% non-fat dry milk at room temperature for 1 h. (l) Repeat step j (m) Incubate the membrane with a goat anti-mouse HRP-conjugated secondary antibody in 1:4000 dilution in TBST containing 0.5% non-fat dry milk at room temperature for 1 h. (n) Repeat step j (o) Mix equal volumes of a luminol enhancer solution and a peroxide solution (e.g., Detection Reagents 1 and 2 from the Pierce ECL western blotting substrate kit). (p) Wet the membrane with the mixture and incubate at room temperature for 2 min. (q) Visualize the proteins using a western blot imaging system, such as ChemiDoc-It2 from UVP. (r) This process should result in the appearance of two bands: an upper band of ~25 kDa corresponding to the IC-peptide-IN-CBD precursor and a lower band of ~20 kDa corresponding to the processed IN-CBD product (see Notes 27–29) (Fig. 5).

Fig. 5 Western blot analysis of seven individual clones from a constructed cyclic oligopeptide library using the anti-CBD antibody, demonstrating that individual clones can exhibit variable levels of expression and intein processing. Lanes 3 and 6 correspond to clones that do not express a full-length IC-peptide-IN-CBD fusion due to the presence of a stop codon and frame-shift, respectively

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6. Deep sequencing analysis of the constructed pSICLOPPS vector: (a) Digest an appropriate amount of the constructed pSICLOPPS library using NcoI and BsrGI in NEBuffer 2.1. for 2 h at 37 oC. This will result in a ~250 bp product containing the variable peptide-encoding region. (b) Prepare the barcoded library using a PCR-free Illuminacompatible library preparation kit, such as the ClaSeek kit from Thermo Scientific, and according to the manufacturer’s instructions (see Note 30). (c) Assess the quality of the barcoded library before proceeding to deep sequencing by quantifying the generated DNA library and verifying its size distribution. In both cases, use commercially available kits, such as the qPCR NGS library quantification kit and the high-sensitivity DNA kit from Agilent Technologies, according to the manufacturer’s instructions. (d) Perform next-generation sequencing of the prepared DNA library using an Ion Torrent high-throughput sequencing platform. (e) Align the ion proton reads to the pARCBD-p sequence using a read aligner, such as bowtie (v2.2.8). (f) Extract the mismatching insert sequences together with their read counts, and cluster the results using the CD-HIT tool. (g) Translate the DNA insert sequences, and consolidate the resulting peptide sequences (see Note 31). (h) Estimate the number of unique peptide sequences encoded by the constructed pSICLOPPS library. (i) Identify possible biases present in the constructed peptide library by analyzing the derived DNA and peptide sequences (see Note 32) 3.3 Monitoring Protein Misfolding and Aggregation in E. coli

When studying a new MisP, folded variants (FolP) should be utilized in order to confirm that protein misfolding can be monitored successfully. If such folded variants are not available, other conditions that affect protein misfolding should be tested instead (see Note 33). 1. Transform E. coli cells with the appropriate vector that enables the production of the MisP-GFP fusion (see Notes 2 and 34). 2. Inoculate triplicates of overnight liquid LB cultures containing the appropriate antibiotic (100 μg/mL Amp or 50 μg/mL Kan) with single bacterial colonies, and grow cells for ~16 h at 37 oC with shaking (see Note 35).

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3. The following day prepare a 1% subculture in fresh LB supplemented with the appropriate antibiotic and incubate at 37 oC with shaking until an OD600 of 0.3–0.5. 4. Induce MisP-GFP production by adding 0.1 mM IPTG or 0.2 μg/mL aTc, and incubate at 37 oC with shaking for a further 2–3 h (see Note 33). 5. Harvest cells corresponding to 1 mL of each culture with OD600 ¼ 1 (for fluorescence measurements) and OD600 ¼ 3 (for SDS-PAGE/western blot analysis) by centrifugation at 6000
g for 2 min. 6. Measure bacterial fluorescence (see Note 36) using: (a) 96-well plate reader l Resuspend the cell pellet in 100 μL of PBS. l

Transfer the cell suspension to a black 96 well-plate (e.g., FLUOTRAC 200 from Greiner).

l

Measure the bacterial cell fluorescence at 510 nm after excitation at 488 nm.

(b) Flow cytometer equipped with a 488 nm solid state laser for excitation and a 530/30 nm band pass filter for detection l

Resuspend the cell pellet in sheath fluid to a final concentration of 105–106 cells/mL.

l

Record a total of 50,000 events using a slow flow rate of approximately 4000–6000 events/s.

l

Gate cells in a forward scatter (FSC) vs. side scatter (SSC) plot in order to eliminate non-cellular events.

l

Plot the gated population in a histogram of fluorescence intensity (FITC) vs. counts and estimate the median fluorescence of each sample.

7. Perform SDS-PAGE/western blot analysis of the total soluble and insoluble protein fractions as described in step 5 from Subheading 3.2 (see Notes 37 and 38). For protein detection, use either a mouse anti-GFP primary antibody in 1:20,000 dilution in TBST or a protein-specific antibody according to the manufacturer’s instructions. 3.4 High-Throughput Screening for Rescuers of Protein Misfolding and Aggregation

Before performing the following tasks, it is important to determine the optimal expression conditions of the system that will result in low cellular toxicity and sufficient production of both the tetrapartite intein fusion and the MisP-GFP fusion (see Note 39). 1. Prepare electrocompetent E. coli cells that carry a MisP-GFPproducing vector, and estimate their transformation efficiency using the pARCBD-p vector (see Notes 1, 2, and 4).

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2. Transform these cells with the constructed pSICLOPPS library, and plate on LB agar supplemented with 34 μg/mL Cm and the antibiotic of the MisP-GFP-producing vector (see Notes 21 and 34). 3. Detach the cells from the agar by scrapping, and wash each plate thoroughly with 2
YT. 4. Collect and vortex the cell suspension in order to break any bacterial clumps. 5. Prepare a sub-culture of an OD600 ¼ 0.1 containing the appropriate antibiotics and arabinose in order to induce the expression of the tetra-partite intein fusion (see Notes 22 and 39). 6. Incubate at an appropriate temperature until an OD600 of 0.3–0.5 at which point initiate the production of the MisPGFP fusion by adding the appropriate inducer (see Note 39). 7. After protein production, dilute an appropriate amount of the cell culture in PBS to a final concentration of 105– 106 cells/mL. 8. Record a total of 50,000 events using a slow flow rate of approximately 4000–6000 events/s. 9. Gate cells in a forward scatter (FSC-A) vs. side scatter (SSC-A) plot in order to eliminate non-cellular events. 10. Plot the gated population in a histogram of fluorescence intensity (FITC-A) vs. counts, estimate the median fluorescence, and select the cell population exhibiting the top 1–2% of fluorescence for sorting. 11. Sort at least ten times more cells than the theoretical diversity of the library using a slow flow rate and harvest the cells into 2
YT medium (see Notes 40 and 41). 12. Plate the collected cells onto LB agar supplemented with the appropriate antibiotics. 13. Repeat steps 3–12 until the median fluorescence of the sorted population ceases to increase. 14. Isolate the plasmid DNA from the last sorted population that exhibited an increase of bacterial fluorescence. 3.5 Evaluation of the Misfolding-Rescuing Ability of the Isolated pSICLOPPS Sub-library

1. Verify the ability of individual members from the pSICLOPPS sub-library to rescue the misfolding of the protein of interest. (a) Transform fresh E. coli cells that carry the MisP-GFPproducing vector with (1) the sorted pSICLOPPS sub-library or (2) the initial pSICLOPPS library. (b) Select a number of individual colonies from the sorted pSICLOPPS sub-library as well as at least two random colonies from the initial pSICLOPPS library that will act as negative controls.

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(c) Inoculate overnight liquid LB cultures containing the appropriate antibiotics, and grow cells for ~16 h at 37 oC with shaking. (d) Prepare a 1% subculture in fresh LB supplemented with arabinose and the appropriate antibiotics, and incubate until an OD600 of 0.3–0.5. (e) Initiate the overexpression of the MisP-GFP fusion by adding the appropriate inducer, and after protein production, measure the fluorescence of the individual cultures using either a plate reader or a flow cytometer as in step 6 of Subheading 3.3. (f) For the cultures that exhibit increased fluorescence compared to the negative control, perform SDS-PAGE/western blot analysis to: l

Verify the production of the tetra-partite IC-peptideIN-CBD fusion and its subsequent processing as described in step 5 from Subheading 3.2.

l

Verify the enhancement of the MisP-GFP solubility as described in step 7 from Subheading 3.3.

(g) Isolate the individual pSICLOPPS members from the sub-library that demonstrate the desirable positive effects. (h) Repeat using triplicate colonies from each individual positive pSICLOPPS sub-library member. 2. When searching for cyclic peptide rescuers of protein misfolding and aggregation, it is recommended to assess whether the positive effects of the selected pSICLOPPS clones are dependent on the splicing activity of the intein, using the Ssp DnaE splicing-deficient variant H24L/F26A [27]. (a) Construct an auxiliary vector, such as pSICLOPPS (H24L/F26A)KanR. l

Prepare and run a PCR reaction according to the manufacturer’s instructions using the pARCBD-p vector as a template, the pARCBDseqFor forward primer, and a reverse primer containing the appropriate gene substitutions for the H24L/F26A mutations (SspDnaE (H24L/F26A)rev). Purify the PCR product using a PCR purification kit.

l

Digest the purified PCR product with NcoI and BglI, the pSICLOPPSKan vector with BglI and HindIII, and the pARCBD-p vector with NcoI and HindIII according to the manufacturer’s instructions.

l

Perform three-way ligation of the above digested products using a T4 DNA ligase to construct the pSICLOPPS(H24L/F26A)Kan vector.

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Transform E. coli cells with the newly constructed pSICLOPPS(H24L/F26A)Kan, and purify the plasmid using a plasmid purification kit.

(b) Construct splicing deficient variants of the selected positive hits from the sorted pSICLOPPS sub-library. l

Digest the auxiliary pSICLOPPS(H24L/F26A) vector and the selected pSICLOPPS positive hits with BglI and HindIII according to the manufacturer’s instructions.

l

Construct the respective pSICLOPPS(H24L/F26A) variants of the positive clones by ligation using a T4 DNA ligase and according to the manufacturer’s instructions.

l

Transform E. coli cells with the newly constructed vectors, and purify using a plasmid purification kit.

l

Verify the presence of the correct insert and the inability to perform protein splicing/peptide cyclization as described in steps 3–5 from Subheading 3.2.

(c) Transform fresh E. coli cells that carry the MisP-GFPproducing vector with (1) either one of the two randomly selected clones from the initial pSICLOPPS library (negative controls), (2) the selected pSICLOPPS positive hits (positive controls), or (3) their pSICLOPPS(H24L/ F26A) variants. (d) Perform protein overexpression and analysis as in Subheading 3.5 using triplicate colonies for each sample. 3. Assess whether the positive effect of the selected pSICLOPPS clones are protein-specific. (a) Replace the MisP under investigation with other non-related misfolded-prone proteins (for a list see, for example, [1]), and evaluate the ability of the selected pSICLOPPS positive hits to rescue their misfolding as described in step 1. 4. Perform DNA sequencing of the selected positive hits, and produce the encoded cyclic peptides by chemical synthesis. 5. Verify the ability of the selected synthetic cyclic peptides to rescue the misfolding of the MisP of interest using wellestablished techniques (e.g., thioflavin-T or Congo red staining, aggregate characterization by transmission electron microscopy), in vitro mammalian cell assays, and, if available, small animal models, such as in Caenorhabditis elegans (see Note 42).

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3.6 High-Throughput Analysis of the Selected pSICLOPPS Sub-library

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If the evaluation performed in Subheading 3.5 resulted in the identification of multiple peptide sequences exhibiting the desirable effects, it is recommended to perform next-generation sequencing of the pSICLOPPS sub-library in order to identify the entire ensemble of potentially bioactive cyclic peptides. 1. Prepare the selected pSICLOPPS sub-library, and perform deep sequencing analysis as described in step 6 of Subheading 3.2. 2. Determine the bioactivity threshold by selecting a number of cyclic peptides appearing in the sorted pool with low frequencies and testing them as described in Subheading 3.5. 3. Design all the circular permutants of the selected cyclic peptides that exceed the identified bioactivity threshold (see Note 43). 4. Enter the peptide sequences into the IEDB clustering tool (http://tools.iedb.org/cluster/) [44], select the desirable similarity threshold, and perform sequence clustering using the fully interconnected clusters (cliques) method (see Notes 44 and 45). 5. Re-integrate the circular permutants into their original cyclic peptide sequence. 6. Plot an undirected network graph using an appropriate visualization software, such as the open-source Gephi software [45]. Represent each distinct cyclic peptide with one node, and connect all cyclic peptides that belong in the same clique with edges. 7. Identify the formed clusters using an appropriate hierarchical method, such as the Girvan-Newman algorithm [49]. 8. Analyze the cyclic peptides from each clique using heat maps in order to identify the distribution of amino acids at each peptide position.

3.7 Identify Structure–Activity Relationships of the Selected Positive Hits

One important advantage of utilizing the microbial discovery platform described herein is that it allows the generation of structure– activity relationships by point mutagenesis in a highly efficient and affordable manner. 1. Construct nucleophile substitution and alanine-scanning variants of the positive hits from the pSICLOPPS sub-library. (a) Design forward mutagenesis primers for: l

Substituting the nucleophile (Cys, Ser or Thr) that is present at position 1 with the other two nucleophiles.

l

Substituting the amino acids at the rest of the positions with alanine (see Note 46).

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(b) Prepare and run PCR reactions according to the manufacturer’s instructions using the selected pSICLOPPS positive hit as a template, each one of the above-designed forward primers and the reverse CBD primer. Purify the PCR products using a PCR purification kit. (c) Digest the purified PCR products and the pSICLOPPSKan vector with BglI and HindIII according to the manufacturer’s instructions. (d) Construct the selected pSICLOPPS variants by ligating the mutated DNA inserts and the pSICLOPPS vector using a T4 DNA ligase and according to the manufacturer’s instructions. (e) Transform E. coli cells with the newly constructed variants, and purify the plasmids using a plasmid purification kit. 2. Evaluate the ability of the constructed variants to rescue the misfolding of the MisP under investigation as described in Subheading 3.5. 3. Identify the sequence motif responsible for high bioactivity by combining the nucleophile and alanine-scanning mutagenesis results and the heat map analysis results from Subheading 3.6 (see Note 47).

4

Notes 1. In order to construct large SICLOPPS libraries with more than 107 members, the transformation efficiency of the electrocompetent cells used should be at least 109 cfu/μg. This should be ideally measured using the pARCBD-p plasmid and after no more than 1 h recovery at 37 oC. Protocols for the preparation of highly efficient electrocompetent cells can be found in the literature [50–52]. 2. If the MisP-GFP fusion is under the control of a T7 promoter, cells that carry the λDE3 lysogen and thus express the T7 RNA polymerase should be utilized (e.g., BL21(DE3) or Tuner (DE3)). If the MisP under investigation contains disulfide bridges, Origami 2(DE3) cells may be utilized in order to provide an oxidizing cytoplasmic environment and promote proper protein folding. 3. For the construction of the SICLOPPS library, we have utilized the Synechocystis sp PCC6803 DnaE intein (Ssp DnaE) due to its low toxicity compared to other novel inteins [53]. However, other inteins may also be used successfully.

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4. We have found that for successfully monitoring protein misfolding and aggregation, a vector that enables the strong expression of MisP-GFP fusions should be used, such as pET-28 a(+) or pASK75 [54]. 5. The MisP of interest should be fused to the GFP reporter via a short flexible linker, such as GSAGSAAGSGEF, that lacks bulky hydrophobic residues or stretches of homologous repeats [55, 56]. 6. For more efficient monitoring of protein folding, a GFP variant containing the F64L and S65T mutations (enhanced GFP, EGFP) derived by Waldo et al. [55] can be used instead of wild-type GFP. 7. These primers enable the construction of combinatorial libraries of random and unbiased head-to-tail cyclic oligopeptides with the general formula cyclo-NuX1X2-Xn, where Nu is a nucleophilic amino acid, essential for the cyclization reaction to occur, i.e., a cysteine (encoded by the TGC codon), a serine (encoded by the AGC codon), or a threonine (encoded by the ACC codon), X is any one of the 20 natural amino acids and n is any number equal or greater than 3. Libraries of different designs (e.g., containing specific amino acids at certain positions or specific peptide tags) can be constructed in the same manner by designing and using the appropriate primers. 8. We have found that by incorporating the first codon of the peptide-encoding region (i.e., TGC, AGC, and ACC for the Cys, Ser, or Thr sub-libraries, respectively) into the zipper primer, the number of frameshifts in the final PCR products is significantly decreased. 9. Special precautions should be taken when handling and disposing EtBr and EtBr-stained gels as it is a known carcinogen. 10. Any available gene can be amplified and inserted into pARCBD-p and in place of KanR (~820 bp). However, in order to enable the facile estimation of the cloning success rate, the size of such gene should be considerably different to the size of the SICLOPPS library PCR products (~560 bp). 11. Ensure that an adequate amount of forward primer is used for the PCR reaction in order to cover the theoretical diversity of the library of interest by more than ten times. Adjust the volume of the PCR reaction accordingly. 12. If the high-fidelity version of HindIII (HindIII-HF) is used, DNA digestion should be performed sequentially, as BglI and HindIII-HF are not compatible in any of the new NEBuffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart); i.e. they don’t exhibit >50% activity in the same buffer. In contrast, BglI and HindIII exhibit 100% and 50% activity in NEBuffer 3.1, respectively,

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and therefore may be used in a double digestion reaction. In this case, however, the duration of the digestion reaction should be limited in order to avoid the star activity of the non-high-fidelity HindIII. We find that 5 h of digestion at 37 oC results in complete digestion and limited star activity. 13. Heat inactivation of enzymes (restriction enzymes, alkaline phosphatase, and T4 DNA ligase) has been found to significantly increase the transformation efficiency and is thus highly recommended after each reaction. 14. The ligation reaction conditions and insert:vector ratios should be optimized whenever a new batch of purified DNA is used as the DNA purity may have a significant impact on the ligation efficiency. We have found a 12:1 insert:vector ratio at 16 oC for 4 h to result in improved ligation efficiency. 15. Desalting of the ligation reactions is very important as salts may cause arcing during electroporation and significantly decrease the transformation efficiency. Another method of desalting is using dialysis filter paper [46]; however, we have found that the use of a purification kit results in comparable transformation efficiencies and is less time-consuming and technical. 16. For in-house electrocompetent cells, the transformation efficiency is increased by producing the cells on the same day that they are used and avoiding freezing and thawing cycles. 17. When transferring the ligation mixture into the electroporation cuvette, the introduction of air bubbles in between the cuvette’s electrodes should be avoided, otherwise resulting in arcing. 18. Residual moisture on the cuvette’s electrodes will result in arcing and failure of the electroporation process. 19. We have found that SOB medium offers a higher electroporation efficiency compared to the commonly used super optimal broth with catabolite repression (SOC). 20. The recovery medium should be added immediately after electroporation as any delay reduces the transformation efficiency dramatically [57]. For higher efficiency, the recovery medium should be pre-heated at 37 oC. 21. Uneven plating will result in library biases as larger colonies will be over-represented in the final mixture. 22. The volume of the cell suspension used for subculturing should cover the theoretical diversity of the pSICLOPPS library by at least ten times. The volume of the subculture should be adjusted accordingly in order to obtain an OD600 of 0.1. 23. For colony PCR experiments, the use of the standard Taq DNA polymerase is recommended due to its higher amplification efficiency compared to other high-fidelity DNA polymerases.

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24. We highly recommend using backbone-specific primers which demonstrate if the insert is of correct size and if it is properly localized within the vector backbone. By utilizing the pARCBDseqFor and p15AoriseqRev primers, a band of ~880 bp indicates successful insertion of PCR library product into the pARCBD-p vector (in place of the KanR gene), while a band of ~1150 bp indicates reclosing of the initial pSICLOPPSKan vector. Alternatively, insert-specific primers can also be utilized, such as the appropriate zipper forward and the CBD reverse primers. 25. Instead of using 2 μL of overnight liquid culture as a template, the selected colony can also be picked using a pipette tip and dissolved into the PCR reaction by mild pipetting. 26. After blocking with non-fat dry milk and incubation with each antibody dilution, membranes should be thoroughly washed with TBST. We recommend washing for 10 min at least three times with fresh TBST. 27. The absence of both the upper and the lower weight bands could indicate one of three things: (1) unsuccessful cloning of the pSICLOPPS library (i.e., the IC-Kan construct is produced instead of the IC-peptide-IN-CBD fusion), (2) presence of a stop codon on the peptide random sequence, or (3) frameshift mutation that hinders the expressing of the full-length tetrapartite fusion. 28. It is expected that different clones will yield variable amounts of the lower molecular weight band, as the intein splicing efficiency is highly dependent on the composition of the intervening peptide sequence. 29. It should be emphasized that the appearance of the lower ~20 kDa band is only an indication of successful intein splicing and does not provide evidence of successful cyclic peptide formation. 30. If possible, it is highly recommended to use a PCR-free method for preparing the samples for deep sequencing, as PCR will unequivocally produce more bias to the library under investigation. 31. Quality assessment of the derived sequencing data should be performed before analyzing the sequencing results. Specifically, DNA sequences that do not start with TGC, AGC, or ACC and that do not correspond to the NNS motif should be excluded from the analysis as they are artifacts produced by misaligning and other errors during the sequencing reaction and initial analysis. 32. Biases in the pSICLOPPS library can appear at both the DNA and peptide levels, so the analysis of both types of sequences should be performed. Biases at the DNA level can be identified

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easily by comparing the reads corresponding to each sequence. Biases at the amino acid level can be identified by plotting heat maps, illustrating the distribution of each amino acid at each position of the peptide encoding region. 33. A range of different inducer concentrations, incubation temperatures, and incubation periods should be tested in order to determine the optimal expression conditions for monitoring the protein misfolding of different MisP-GFP constructs. We have found that these vary greatly depending on the protein under investigation. 34. Freshly transformed E. coli cells should be used for the protein production experiments. 35. Experiments should be performed at least in triplicate in order to ensure reproducibility and identify potential outliers. 36. Fluorescence measurements of E. coli cells overexpressing MisP-GFP fusions should be performed within 1 h after collection. 37. The insoluble fraction is prepared by adding the same volume of PBS to the cell pellet derived after the centrifugation of step 5e from Subheading 3.2. 38. The soluble levels of MisP-GFP should be significantly decreased compared to the soluble FolP-GFP levels (if available) or compared to MisP-GFP protein produced under conditions that enhance protein solubility and promote proper folding. 39. Overexpression of different members of the pSICLOPPS library may result in variable levels of cellular toxicity. Therefore, plating assays should be performed in order to determine the inducer concentrations and incubation temperatures that exhibit decreased toxicity, impeding the introduction of growth-based bias, and sufficient production of the respective protein fusions. 40. A slow flow rate offers greater resolution and accuracy. However, when sorting larger libraries it is advisable to increase the flow rate in order to perform FACS sorting in a reasonable timeframe. 41. We have found that collecting the E. coli cells in 2
YT medium significantly increases their viability after FACS sorting. 42. It is important to note that the bacterial system only provides strong indications of the misfolding-rescuing activity of the selected sequences and not solid evidence. Therefore, these effects should be verified by performing in vitro and in vivo aggregation and functional assays using synthetic cyclic peptides of high purity.

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43. As amino acid alignment is performed using linear peptide sequences, all circular permutants should be taken into consideration in order to portray the cyclic nature of the selected peptides. As an example, the circular permutants of cyclic peptide cyclo-ABCD are the liner sequences: ABCD, BCDA, CDAB, and DABC. 44. We have found that a similarity threshold of 60–70% is suitable for grouping our identified cyclic peptides into distinct and well-defined clusters. However, this threshold is dependent on the size of the cyclic peptides under investigation and the efficiency of the selection process during FACS sorting. 45. The fully interconnected clusters method allows all peptides in a clique to share the specified level of homology, but at the same time, one peptide can be part of multiple cliques. In this way, the relationships between the selected cyclic peptides are illustrated in more detail. 46. As it is essential for the first amino acid of the peptide extein to be a nucleophile (Cys, Ser, or Thr), substitution of the amino acid at position 1 with alanine would hinder the production of the cyclic peptide. 47. The amino acids that are responsible for the bioactivity of the selected positive hit are revealed by the alanine substitutions that result in decreased bioactivity. However, using the data from the deep sequencing analysis, further mutagenesis experiments should be performed in order to define in more detail the bioactive sequence motifs. 48. Using this technology, we have constructed libraries of cyclic tetra-, penta-, hexa-, and heptapeptides (n ¼ 3, 4, 5, and 6 respectively); however, libraries of larger peptides can also be constructed.

Acknowledgments This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Project “ProMiDis”; grant agreement no 819934). References 1. Chiti F, Dobson CM (2017) Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annu Rev Biochem 86:27–68 2. Carrell RW, Lomas DA (1997) Conformational disease. Lancet 350(9071):134–138

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Chapter 14 Site-Specific Incorporation of Non-canonical Amino Acids by Amber Stop Codon Suppression in Escherichia coli Uchralbayar Tugel, Meritxell Galindo Casas, and Birgit Wiltschi Abstract The site-specific incorporation of non-canonical amino acids with reactive side chains is a powerful tool for the directed chemical modification of proteins. Here we provide a protocol for the site-specific incorporation of a lysine-derivative with a bioorthogonal azido group in proteins expressed in Escherichia coli. The successful incorporation is assessed by bioorthogonal conjugation to a fluorescent dye using copper(I)catalyzed and strain-promoted azide-alkyne cycloaddition. Key words Non-canonical amino acid, Stop codon suppression, Site-specific incorporation, Reactive side chain, Bioorthogonal conjugation

1

Introduction In nature, all existing life forms use 20 canonical L-alpha-amino acids (cAAs) as building blocks of their proteins. Nevertheless, many other amino acids occur naturally and they have been found in different plants and microorganisms as a product of secondary metabolism [1, 2]. With the exemption of the 21st and 22nd amino acids selenocysteine [3] and pyrrolysine [4], these non-canonical amino acids (ncAAs) are usually not incorporated into proteins by ribosomal translation. NcAAs are desirable building blocks for protein engineering because they showcase an extraordinary, rich structural and functional repertoire. Among others, ncAAs with uniquely reactive side chains are particularly attractive as they facilitate bioorthogonal conjugation reactions [5, 6], for instance, to install artificial post-translational modifications [7–14]. Consequently, techniques to make ncAAs available for protein engineering in different hosts were developed [15–17]. Analogs of the cAAs can be incorporated into proteins simply by complementing the appropriate amino acid auxotrophy of the host strain [18, 19]. In this way, the ncAA replaces the

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_14, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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corresponding cAA at all cognate codons. Unless the target sequence contains only a single cognate codon, the site-specific incorporation of the ncAA is not possible in this way. For sitespecific incorporation of ncAAs, translation stop codons have been exploited predominantly [14]. In principle, all three stop codons, ochre (UAA), opal (UGA), and amber (UAG), can be suppressed with ncAAs [20]. The amber codon is often used because it is the least frequent stop codon in many organisms (see, e.g., http://www.kazusa.or.jp/codon/). To incorporate an ncAA at an in-frame amber stop codon, the expression host is equipped with an amber suppressor tRNACUA, which “reads” the amber codon. It is charged with the ncAA by an aminoacyl-tRNA synthetase (aaRS), which is specific for this ncAA. The aaRS/tRNACUA pair must be orthogonal in the expression host: This means that the amber suppressor tRNACUA must not be charged by any of the host aaRSs to avoid the incorporation of a cAA at the in-frame amber codon in the target protein. Likewise, the aaRS must not charge host tRNAs with the ncAA. Most importantly, the aaRS should be specific for the ncAA and should not aminoacylate the tRNACUA with any of the cAAs. The pyrrolysyltRNA synthetase (PylRS) enzymes from Methanosarcina mazei and Methanosarcina barkeri together with their cognate amber suppressor tRNACUA fulfill these criteria: In their natural archaeal hosts, the M. mazei and M. barkeri PylRS/tRNACUA pairs incorporate the 22nd amino acid pyrrolysine at in-frame amber codons of selected proteins [4]. In recombinant hosts such as E. coli [21], S. cerevisiae [22], and mammalian cells [23], they demonstrate a remarkable substrate tolerance. More than 20 (pyrro)lysine derivatives serve as substrates for the native pairs, and twice as many can be incorporated using mutant PylRS/tRNACUA pairs [7, 24]. To furnish a recombinant host with the ability for site-specific incorporation of an ncAA, the orthogonal aaRS/tRNACUA pair is co-expressed with the target protein in the presence of the ncAA. Here we describe the site-specific incorporation of the reactive ncAA, Nε-[(2-azidoethoxy)-carbonyl]-L-lysine (AzK) into a target protein in E. coli and the bioorthogonal labeling of the azidofunctionalized protein. The procedure includes the design of the amber mutant of the target protein and the construction of the co-expression vector for the amber mutant and the MmPylRS/ MmtRNACUA pair; the incorporation of AzK into the target protein; the purification of the target protein via Ni-NTA chromatography and its characterization; and finally the assessment of the incorporation of AzK by bioorthogonal conjugation with a fluorescent dye using copper(I)-catalyzed or strain-promoted azide-alkyne cycloaddition.

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Materials

2.1 Specialized Equipment

1. Electroporator, e.g., MicroPulser Electroporator (Bio-Rad Laboratories, Hercules, CA), or equivalent and electroporation cuvettes, 0.1 cm gap (Bio-Rad Laboratories). 2. Branson Sonifier 250 (Emerson Electric, St. Louis, MO) or similar cell disruption equipment or method. 3. Disposable 10 mL polypropylene columns (Thermo Fisher Scientific, Waltham, MA). 4. Ultrafiltration columns, e.g., VivaSpin 3000 MWCO columns (Sartorius AG, Go¨ttingen, Germany). 5. Thermoshaker, e.g., ThermoMixer® comfort (Eppendorf AG, Hamburg, Germany).

2.2 Reagents and Solutions

1. Use doubly distilled water (ddH2O) for the preparation of all media components and buffers. Common sources for chemicals are Sigma-Aldrich (St. Louis, MO), Carl Roth GmbH (Karlsruhe, Germany), or Merck KGaA (Darmstadt, Germany). 2. E. coli strains: BL21(DE3), E. coli B F
ompT hsdSB(rB
mB
) dcm+ gal λ(DE3) (Agilent Technologies, Santa Clara, CA); Top10F0, E. coli K-12 F0 [lacIq Tn10(Tetr)] mcrA Δ(mrrhsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(araABC-leu)7697 galU galK rpsL(Strr) endA1 λ
(Thermo Fisher Scientific). 3. Plasmid: pMmOP-eGFPY40am (see Fig. 1 and Note 1). 4. LBkan selection plates and medium: LB agar (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 15 g/L agar; Carl Roth GmbH) containing 50 mg/L kanamycin (Carl Roth GmbH). LBkan medium is the same as in LBkan selection plates but without agar. 5. Stock solutions for the preparation of M9 medium: 5 M9 salt (240 mM Na2HPO4, 110 mM KH2PO4, 45 mM NaCl, and 95 mM NH4Cl) sterilize by autoclaving. Prepare 1 M MgSO4 and 1 mg/mL CaCl2 stock solutions and autoclave. Prepare 1 M glucose and filter through 0.22 μm membrane filter (Merck KGaA); do not autoclave. The composition of the trace element stock solution is indicated in Table 1. These components are stored at room temperature. A 1 mg/mL thiamine stock solution is filter-sterilized using a 0.22 μm membrane filter. For 50 mL of 1 mg/mL biotin stock solution, dissolve 50 mg biotin in 45 mL ddH2O. Add small aliquots of 1 N NaOH until the biotin has dissolved and fill up to the final volume with ddH2O. Filter-sterilize using a 0.22 μm membrane filter. The thiamine and biotin stock solutions are stored at 4  C.

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Fig. 1 Vector map of pMmOP-eGFPY40am. The pyrrolysyl-tRNA synthetase from M. mazei (MmPylRS) is under the control of the arabinose-inducible PBAD promoter. The expression of the amber suppressor MmtRNACUAPyl from M. mazei is controlled by the proK promoter (PproK) and terminator (TproK). The target protein eGFP Y40am is expressed from the IPTG-inducible T5 promoter (PT5-lacO). Additional parts of the pMmOP plasmid: araC regulator of PBAD, ParaC promoter of araC, p15a ori medium copy origin of replication, neokan kanamycin resistance marker with Pneokan promoter, rrnBT1, rrnBT2, rrnC, λt0 are terminator sequences. C_fwd (50 -ATTTAAATCTCGAGAAATCATAAAAAATTTATTTGC-30 ), C_rev (50 -GAGGTCATTACTGGATCTATCAACAG-30 ), primers for control PCR and sequencing.

6. M9 medium is prepared freshly from the stock solutions as outlined in Table 2. For M9kan medium, add 50 mg/L kanamycin. Kanamycin is sterile-filtered with a 0.22 μm membrane filter before adding it to the medium. 7. Incorporation of AzK: 20% (w/v) L(+)-arabinose (Carl Roth GmbH) stock solution for the induction of PylRS and 1 M IPTG stock to induce the expression of the target protein. Both stock solutions are sterilized using a 0.22 μm membrane filter. The arabinose stock is stored at room temperature; store 1 mL aliquots of the IPTG stock at
20  C. The ncAA, e.g., AzK, is added at a final concentration of 1–10 mM. Prepare a

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Table 1 Composition of the trace elements stock solution Compound

CAS No.

MW (g/mol)

FeSO4·7H2O

7782-63-0

278.02

144

MnSO4·H2O

10034-96-5

169.02

59

AlCl3·6H2O

7784-13-6

241.43

41

CoCl2·6H2O

7791-13-1

237.90

31

ZnSO4·7H2O

7446-20-0

287.54

7

Na2MoO4·2H2O

10102-40-6

241.95

8

CuCl2·2H2O

10125-13-0

170.48

6

H3BO3

10043-35-3

61.84

8

7647-01-0

36.46

5000

HCl, fuming

a

Concentration (mM)

The components are dissolved in HCl diluted 1:1 with ddH2O and filled up to 1 L with ddH2O. The trace elements stock can be stored at room temperature. Source of chemicals, e.g., Merck KGaA a 37%, density 1.19 g/mL

50–100 mM stock of AzK in 10 mM sodium phosphate (NaPi) buffer pH 8.0 ([25]; see Note 2) and filter-sterilize. Amino acid solution should not be stored; prepare always fresh. 0.9% (w/v) NaCl solution to wash the cells after harvest. 8. Stock solutions for protein purification: 1 M NaH2PO4; 1 M Na2HPO4, and 5 M NaCl. The stocks can be stored at room temperature without sterilizing. 9. Lysis, wash, elution, and cleaning buffers for protein purification: 50 mM NaPi, 150 mM NaCl, 10–500 mM imidazole as outlined below, pH 7.2. Dissolve the imidazole in 600 mL ddH2O and add 11.1 mL of 1 M NaH2PO4, 38.9 mL of 1 M Na2HPO4 and 30 mL 5 M NaCl. Fill up to 900 mL with ddH2O and adjust the pH to 7.2 with HCl, then fill up to 1 L with ddH2O and store at 4  C with light protection. Imidazole concentrations: Lysis buffer, 10 mM; wash buffer, 30 mM; elution buffer, 300 mM; cleaning buffer, 500 mM. 10. Ni-NTA agarose beads (Qiagen, Hilden, Germany) for the purification of the hexahistidine-tagged target protein. 11. PBS for protein storage: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4. 12. CuAAC reagents: Prepare protein solution in 50 mM NaPi buffer pH 7.0 [25]; 100 mM sodium ascorbate, prepare fresh; 10 mM CuSO4, store at 4  C; 50 mM tris-3-hydroxypropyltriazolyl-methyl-amine (THPTA; Sigma-Aldrich), store at 4  C; 50 mM ethylenediaminetetraacetic acid (EDTA), store at
20  C. Sulfo-cyanine-3-alkyne dye (Lumiprobe GmbH,

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Table 2 Preparation of 1 L M9 medium from stock solutions Stock solution

Volume

Final concentration

5X M9 salt 240 mM Na2HPO4 110 mM KH2PO4 45 mM NaCl 95 mM NH4Cl

200 mL

1X 48 mM 22 mM 9 mM 19 mM

Trace elements FeSO4·7H2O MnSO4·H2O AlCl3·6H2O CoCl2·6H2O ZnSO4·7H2O Na2MoO4·2H2O CuCl2·2H2O H3BO3 HCl 1 M MgSO4

60 μL

1 mL

8.6 μM 3.5 μM 2.5 μM 1.8 μM 0.4 μM 0.5 μM 0.4 μM 0.5 μM 300.0 μM 1 mM

1 mg/mL CaCl2

1 mL

1 mg/L

1 mg/mL thiamine

1 mL

1 mg/L

1 mg/mL biotin

1 mL

1 mg/L

1 M glucose

20 mL

20 mM

Hannover, Germany) is light-sensitive; dye solutions should be stored in black polypropylene tubes at 4  C. 13. SpAAC reagents: sulfo-Cyanine3 DBCO dye (Cy3-DBCO; Lumiprobe). The DBCO moiety reacts spontaneously with an azide group to produce a stable triazole, which is referred to as Cu (I)-free or strain-promoted click reaction (SpAAC). The dye is light-sensitive, store dye solutions in black polypropylene tubes at 4  C. 14. SDS-PAGE: Use commercially available pre-cast SDS gels in combination with commercial buffers, e.g., NuPAGE (Thermo Fisher Scientific), or prepare custom SDS gels and buffers as described previously [26–28]. 5 SDS sample buffer (250 mM Tris/Cl pH 6.8, 10% (w/v) SDS, 50% (v/v) glycerol, 0.5 M dithiothreitol (DTT), 0.2% (w/v) bromophenol blue); dilute 1:5 with ddH2O to obtain 1 SDS sample buffer; destaining solution (7.5% (v/v) acetic acid, 20% (v/v) EtOH); the Coomassie stain is destaining solution containing 2.5 g/L Coomassie® Brilliant Blue G 250.

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3

273

Methods

3.1 Design and Preparation of the Amber Mutant

1. Choose the position for the incorporation of AzK in the target protein of interest. If AzK replaces a specific residue, e.g., in the active center of the target protein, the codon for the specific residue is simply exchanged for the amber codon TAG in the DNA coding sequence. It should be considered that the incorporation efficiency of the ncAA as well as its productive use for bioorthogonal conjugation is dependent on several conditions: (1) The azido-group should be solvent exposed so that it is accessible for bioorthogonal conjugation with small or large molecules. (2) Neither the incorporation of AzK nor its bioorthogonal conjugation should interfere with protein folding, stability, or function (unless this is desired). (3) The incorporation efficiency of the ncAA depends on the sequencecontext of the in-frame amber codon on the mRNA [29, 30] and on the structure of the ncAA [31]. 2. If the 3D structure of your protein of interest is available, choose the incorporation position using structure imaging software such as PyMOL [32] or YASARA [33, 34]. Since it is difficult to predict the incorporation efficiency of the ncAA at a specific position in the protein, it is judicious to select several incorporation positions. If the 3D structure of the target protein is not available, the comparison of the amino acid with known homologs or homology modeling can aid in making an educated guess about suitable incorporation positions. In this case, be sure to select several positions to test. 3. The reporter protein eGFP Y40am on plasmid pMmOPeGFPY40am (Fig. 1) carries the amber mutation at position Y40. This site has been shown to be permissive for the incorporation of ncAAs [35, 36], and it is located upstream of the fluorophore (positions T65-Y66-G67). Consequently, the fluorescence readout is directly correlated to the incorporation efficiency of ncAAs. 4. Fusion tags [37] greatly facilitate the purification of the target protein. If possible, the purification tag should be fused to the C-terminus of the protein. The tag is only produced upon readthrough at the in-frame amber codon; consequently, only fulllength protein products, which contain the ncAA, are purified. Here, we describe the incorporation of AzK into a hexahistidine-tagged target protein (see Note 3) because the hexahistidine-tag is frequently used to purify recombinant proteins from E. coli. 5. The in-frame amber codon TAG can be introduced into the coding DNA sequence of the gene of interest (GOIam) using QuikChange PCR [38, 39], overlap extension PCR [40], or gene synthesis.

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3.2 Cloning of the Amber Mutant into pMmOP

1. Excise the eGFP Y40am insert from pMmOP-eGFPY40am using BglII (see Fig. 1). 2. Insert the gene of interest (GOIam) by Gibson assembly [41]; alternatively, restriction cloning may be used (see Note 4). 3. Amplify the resulting plasmid pMmOP-GOIam in a suitable E. coli strain, e.g., Top10 F0 and select kanamycin-resistant clones on LBkan selection plates. 4. Analyze the selected clones by colony PCR [42] using primers C_fwd and C_rev (see Fig. 1). 5. Isolate pMmOP-GOIam from positive clone(s) using a commercial plasmid isolation kit, e.g., GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific), and sequence verify (see Note 5).

3.3 Site-Specific Incorporation of AzK into the Target Protein in E. coli

1. Transform the expression strain of choice, e.g., E. coli BL21 (DE3) (see Note 6), with pMmOP-GOIam using electroporation [43] or a similar transformation method [44] and plate on LBkan selection plates. To maintain the strain BL21(DE3) {pMmOP-GOIam}, prepare a glycerol stock [45] from a single kanamycin-resistant colony. 2. Prepare an overnight culture (ONC) by inoculating 10 mL LBkan or M9kan medium in a sterile 50 mL disposable plastic tube with a single colony of the E. coli strain BL21(DE3) {pMmOP-GOIam}, and incubate overnight (~16 h) at 37  C with vigorous shaking (see Note 7). 3. Determine the cell density of the ONC by spectrophotometric measurement of the attenuance at 600 nm (D600; [46]). Harvest enough culture volume so that the main culture (see Note 8) can be inoculated to a start D600 of 0.1 If the ONC was prepared in LBkan medium, wash the cells 1–2 times with 10 mL M9kan medium. Inoculate the main culture to a start D600 of 0.1 (see Note 9). 4. Incubate the cells at 37  C with vigorous shaking (see Note 10) and regularly record the D600. 5. When the cells reach a density of D600 0.7–0.8, they are ready for the induction of the target protein expression. Collect a sample equal to 1 D600 of the non-induced cells for analysis by SDS-PAGE. Harvest cells in a table-top centrifuge, discard the medium supernatant, and store the cell pellet at
20  C until use. 6. To the main culture, add 5 mM AzK (see Note 11), as well as 0.5 mM IPTG and 0.2% (w/v) L-(+)-arabinose to induce the expression of the target protein and the MmPylRS. Decrease the incubation temperature to 28  C and incubate overnight (see Note 12).

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7. After overnight incubation, collect a sample of the induced culture equaling 1 D600 and treat as the non-induced sample described in step 5. 8. Harvest the cells by centrifugation at 5000  g at 4  C for 20 min. Discard the medium supernatant, and wash the cell pellet once with the same volume of 0.9% (w/v) NaCl solution. Keep the cells on ice until they are used. Alternatively, the cell pellet can be stored at
20  C for several months. 3.4 Purification and Characterization of the AzK-Labeled Target Protein

1. If a frozen cell pellet is used, thaw it on ice. On ice, resuspend the cell pellet in lysis buffer at a ratio of pellet weight (grams) to buffer (mL) of 1:1 to 1:4 (see Note 13). Disrupt cells by sonication on ice (see Note 14), e.g., using a Branson Sonifier 250 at an output power of 7 and 75% duty cycle, 10 short bursts of 10 s followed by intervals of 30 s for cooling. Mix or shake the sample between bursts to increase sonication efficiency. 2. Remove cell debris by centrifugation at 17,000  g and 4  C for 30 min. After centrifugation, decant the supernatant and use it directly for protein purification. Do not store the cell-free extract (see Note 15). 3. For the purification of the hexahistidine-tagged AzK-labeled target protein by gravity flow Ni-NTA chromatography, prepare a disposable 10 mL polypropylene column. Equilibrate the Ni-NTA beads to ambient temperature; then mix properly and transfer 2 mL beads into the column without introducing air bubbles. 4. Wash the column three times with 35 mL ddH2O each; then equilibrate the column by washing three times with 35 mL lysis buffer. 5. Load the cell-free extract onto the equilibrated column, and collect the flow-through (see Note 16). 6. Wash the column with 20 mL wash buffer to remove unspecifically bound proteins from the column; collect the wash fraction. 7. Elute the target protein ten times with 1 mL elution buffer, and collect each elution fraction. 8. Regenerate the resin with 20 mL cleaning buffer; optional: collect the cleaning fraction. Preserve the column with 20 mL 20% (v/v) ethanol and store it at 4  C. 9. Assess the protein concentration in all collected fractions. The protein concentration can be measured by the Bradford assay [47] or absorption measurement at 280 nm, e.g., using a NanoDrop spectrophotometer (Thermo Fisher Scientific). For analysis by SDS-PAGE [28], prepare 50 μg of protein in

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15 μL 1 SDS sample buffer. Add 50 μL 1 SDS sample buffer to the whole cell samples from steps 5 and 7 in Subheading 3.3 and use 5–10 μL for analysis. Heat all samples at 95  C for 5–10 min and analyze directly by SDS-PAGE or store at
20  C until use. 10. Exchange the buffer of the elution fractions, e.g., for PBS or any other suitable buffer, by ultrafiltration, by dialysis, or with a desalting column (see Note 17). Assess the functional integrity of the AzK-labeled target protein employing a suitable activity assay. 11. To confirm the incorporation of AzK, analyze the mass of the isolated protein, e.g., by intact mass analysis using LC-ESI-MS and peptide sequencing with LC-MS/MS. 3.5 Quick Check of AzK Incorporation by Coupling to a Fluorescent Dye 3.5.1 Copper(I)-Catalyzed Azide-Alkyne Conjugation (CuAAC)

1. In a total volume of 20 μL mix 4 μg isolated protein (the final protein concentration should be 0.2 μg/μL; see Note 18) in 50 mM NaPi buffer pH 7.0 with 0.5 mM CuSO4, 5 mM sodium ascorbate (freshly prepared), and 2.5 mM THPTA. Sodium ascorbate reduces Cu(II) to Cu(I) [48, 49], and THPTA is a Cu(I)-binding ligand that accelerates the reaction [50]. Treat unlabeled wild-type target protein in the same way, and include it as a negative control to assess unspecific binding of the dye. 2. Incubate the reaction mixtures at room temperature with vigorous shaking (e.g., 550 rpm on a thermoshaker) for 1 h. 3. Stop the reaction by adding 5 mM EDTA and 5 μL of 5 SDS sample buffer and heat samples at 95  C for 10 min. 4. Separate the samples on an SDS-gel, and after the completion of the SDS-PAGE, do not strain the gel but remove excess unbound fluorophore by soaking in destaining solution. Expose the washed gel to UV light (302 nm) and record the image. The AzK-labeled target protein should appear as a fluorescent band while the negative wild-type control does not show fluorescence, unless unspecific background binding occurs. Afterward, the protein bands are stained with Coomassie Brillant Blue [26, 51] to assess the purity of the samples and to compare the protein amounts.

3.5.2 Strain-Promoted Azide-Alkyne Conjugation (SpAAC)

1. CuAAC can have negative effects on proteins [52] and cells [53]. Strain-promoted cycloaddition of azides and cyclooctynes provides a mild and copper-free alternative [54]. For SpAAC, mix 1.5–3 mol equivalents of Cy3-DBCO with 1 mol equivalent of azide-containing target protein, e.g., for a single sample for analysis by SDS-PAGE, prepare 4 μg of protein in PBS in a total volume of 15 μL. Since reduced cysteine residues can cross-react with cyclooctynes (see Note 19), an

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additional reaction mix should be prepared with the unlabeled wild-type protein as a negative control. 2. Incubate the reaction mixtures at room temperature with vigorous shaking (e.g., 550 rpm on a thermoshaker) for 1 h. 3. It is not necessary to stop the reaction; the bioconjugation products can be analyzed directly by SDS-PAGE as described for the CuAAC in Subheading 3.5.1.

4

Notes 1. The pMmOP plasmid is available from the authors upon request. The plasmid encodes the wild-type MmPylRS/ MmtRNACUAPyl pair, which can be used for the incorporation of AzK and a number of other (pyrro)lysine derivatives (for a list of structures, see [24]). 2. If an ncAA is not readily dissolved, titration with diluted NaOH or HCl solutions is beneficial. Dissolve very apolar ncAAs at 1 M in DMSO [55]. 3. A hexahistidine-tag is not recommended for proteins with a metal center because the metal can be absorbed by the NTA matrix during Ni-NTA chromatography. Anaerobic conditions reduce the Ni2+ absorbed to the NTA; thus Ni-NTA chromatography is not the purification method of choice for strictly anaerobic proteins [37]. An alternative purification tag should be used in such cases. 4. For restriction cloning, include flanking BglII sites at the 50 and 30 -ends of the target gene sequence, e.g., during gene synthesis or in the forward and reverse primers for PCR amplification. Cut the target DNA sequence and the pMmOP plasmid with BglII, dephosphorylate the cut vector with alkaline phosphatase (Thermo Fisher Scientific) to prevent self-ligation, gel purify the insert and the backbone vector using a commercial gel extraction kit, e.g., GeneJET Gel Extraction Kit (Thermo Fisher Scientific), and ligate the DNA fragments. Since the insert can be ligated in either orientation, we recommend designing specific forward (T_fwd) and reverse (T_rev) primers for the target sequence. Colony PCR with primers T_fwd/C_rev and T_rev/C_fwd (for the priming of C_fwd and C_rev on pMmOP refer to Fig. 1) will indicate not only the presence but also the orientation of the insert. 5. Gibson assembly may introduce undesired mutations; sequencing is therefore mandatory if Gibson assembly is used for plasmid construction.

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6. Expression strains without the λDE3 lysogen, such as BL21 (genotype: E. coli B F
ompT hsdSB(rB
mB
) dcm+ gal; Merck KGaA), are suitable as well since pMmOP does not carry a T7 promoter (see Fig. 1). 7. ONCs that have been stored at 4  C can be re-used for up to 1 week for the inoculation of main cultures. 8. 50–100 mL medium are good for initial trial expressions; 1 L of medium is sufficient for routine expression of most target proteins. However, the volume of the medium depends on the yield of the protein of interest. Complex LB medium can be used for the site-specific incorporation of ncAAs; nevertheless, M9 minimal medium is recommended to avoid interference of excess cAAs. 9. Pre-warming the medium for the main culture decreases the lag phase of bacterial growth. 10. Use culture vessels of appropriate size since aeration of the culture is essential: The ratio of the vessel volume to the culture volume should be at least 5:1, i.e., use a maximum of 200 mL culture in a 1 L Erlenmeyer flask. Use baffled flasks and shake vigorously. 11. Most ncAAs are used at concentrations of 1–10 mM; 5 mM of AzK is a good starting concentration. 12. Overnight induction is good for many proteins; nevertheless, shorter or longer induction periods may be necessary depending on the expression profile of the target protein. 13. Lysis buffer is optimal for the purification of hexahistidinetagged proteins by Ni-NTA chromatography. If the protein of choice requires a different buffer to prevent its functional and/or structural integrity, note that high concentrations of buffer components containing strong electron-donating groups such as NH4+, glycine, arginine, or Tris; strong chelating agents such as EDTA and EGTA; or strong reducing agents, e.g., DTT and ionic detergents, such as SDS, are compatible with the Ni-NTA matrix only to a certain extent [56]. 14. During cell lysis, protein degradation by cellular proteases may occur. To avoid protease degradation, add 0.1–1 mM PMSF or other protease inhibitors. 15. If storage of the cell-free extract cannot be avoided, add protease inhibitor and flash-freeze in liquid nitrogen. 16. For a more efficient purification, mix the beads with the cellfree extract for at least 15 min rotating at 4  C. 17. Proteins may precipitate when stored frozen in elution buffer containing imidazole at high concentrations [57]; in addition imidazole may affect downstream applications such as protein

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concentration determination, NMR, or crystallography [58]. For these reasons, it is recommended to remove imidazole from the elution fractions as soon as possible. 18. For bioorthogonal conjugation, purified target protein with the azido-amino acid incorporated or the cell-free lysate containing the azido-functionalized target protein may be used. Use 4 μg of isolated protein or 40 μg total protein of the cellfree lysate in the conjugation reaction. 19. Reduced thiols of cysteine side chains can cross-react with cyclooctynes. This undesired side reaction can be reduced by adding β-mercaptoethanol to the reaction mixture [59] or by the alkylation of free cysteine-thiols with iodoacetamide [60].

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INDEX A Affibodies....................................................................... 129 Affimer scaffold ............................................................. 129 Affitins............................................................................ 154 to bacteria ................................................................ 166 generation................................................................ 158 from isolated clones ................................................ 166 production ............................................................... 167 ¨ KTAprime plus system ...................................... 228, 232 A Alzheimer’s disease–associated amyloid-β peptide (Aβ42) .......................................................... 241 Amber codon........................................................ 268, 273 Amino acids ................................................................... 263 Ampicillin ...................................................................... 229 Annexin V (AnxV) ......................... 54, 56–58, 60, 62, 63 Antibiotics ..................................................................... 242 Antibody fragment........................................................ 178 Arginine-rich peptides ..........................41, 43, 44, 47, 48 Artificial cofactor ........................................................... 216 Artificial metalloenzymes description ............................................................... 213 formation ................................................................. 214 protein scaffold........................................................ 215 screening effort ....................................................... 214 selectivity ................................................................. 213 variants ..................................................................... 214 Arylthiol MPAA ................................................................ 2 Asp/Asn-Gly dipeptides ................................................. 14 Aspartimide formation amino acid to HMPA ChemMatrix® Resin .......................... 20 to SEA-Trt ChemMatrix® Resin ..................19, 20 Asp/Asn-Gly dipeptides in proteins ........................ 14 by-product formation .........................................13, 25 chemical synthesis, SUMO-2 and SUMO-3................................................. 14 cleavage step ........................................................ 22–23 description ................................................................. 13 elongation, automated peptide synthesizer ...............................................21, 22 Fmoc-Asp(OtBu)[Dmb]Gly-OH, manual coupling ...................................................21, 22 HPLC analysis and purification................................ 17

HPLC purification, SUMO-2/3C and SUMO-2/3N peptides ............................23, 24 isopeptidic structure.................................................. 13 lyophilization ............................................................. 17 MALDI-TOF analysis ............................................... 19 N-terminal peptide segments ................................... 14 organic solvents and chemicals........................... 16–18 oxidation SEAon peptides ......................................... 23 SEA-mediated ligation.............................................. 14 solid-phase peptide synthesis, peptide segments ........................................................ 19 SPPS........................................................................... 14 synthesis, SUMO-2 and SUMO-3 .................... 24–26 TFA ............................................................................ 16 tools ........................................................................... 13 Azide-alkyne coupling .................................................... 40

B Bacteria affinity and specificity .............................................. 154 affitins....................................................................... 166 ELISA ...................................................................... 169 growth ..................................................................... 157 phage display ........................................................... 153 ribosome display selection .................... 157, 163, 164 Bacterial system ............................................................. 262 Binding/internalization assays ..................................... 184 Bioactive peptides ........................................................... 38 Biolayer interferometry (BLI) ...................................... 184 Biomedical engineering .................................................. 37 Bioorthogonal conjugation ................267, 268, 273, 279 Biotin ............................................................................. 214 B4F ....................................... 220, 221, 223, 231, 232 biotinylated cofactor designs .................................. 216 biotinylated cofactors.............................................. 218 derivatization ........................................................... 214 moiety, biotinylated ligands.................................... 214 streptavidin interaction ........................................... 214 Biotin-binding-site-vestibule ........................................ 216 Biotin-4-fluorescein (B4F) ................................. 220, 221, 223, 231, 232 Bis(2-sulfanylethyl)amido (SEA), see SEA ligation Bis-silylated peptide ..................................................80, 84

Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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PEPTIDE

284 Index

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PROTEIN ENGINEERING: FROM CONCEPTS

C Canonical L-alpha-amino acids (cAAs)........................ 267 Carbohydrate antigens.................................................... 54 Cell free extracts (CFE) ....................................... 214, 222 Cell-surface protein....................................................... 153 Chemical controls ........................................................... 64 Chemical synthesis ............................................................ 1 Chemoselective amide bond-forming reactions ...................................................29, 31 Chitosan CuAAC ...................................................................... 41 derivative N3-chitosan polymer................................ 48 high molecular weight chitosan ............................... 42 immobilization, synthesized peptides ...................... 46 peptide-chitosan conjugation .............................42, 43 peptide-N3-chitosan conjugate ................................ 45 polymer’s amines into azides ..............................44, 46 synthesis, peptide-chitosan conjugates .................... 44 ultrathin films ............................................... 42, 45, 46 Chromosomal gene libraries cloning ..................................................................... 190 common kits and reagents...................................... 191 directed evolution ................................................... 190 DNA manipulation ................................................. 190 E. coli (see Escherichia coli, chromosomal gene libraries) oligonucleotides ...................................................... 192 PCR protocols ................................................ 207, 208 in plasmids ............................................................... 190 plasmid libraries....................................................... 190 S. thermophilus (see Streptococcus thermophilus, chromosomal gene libraries) CIS display in vitro selection (see In vitro selection) N-terminal library ................................................... 182 PCR.......................................................................... 183 protein for evolution............................................... 173 protein scaffolds ...................................................... 180 protein–DNA complexes ........................................ 182 selection outcome .......................................... 183–185 target binding screen .............................................. 183 therapeutic applications .......................................... 174 Classical random mutagenesis methods....................... 135 Clustering tool .............................................................. 245 Clusters method ............................................................ 263 Collagen-biomimetic covalent hydrogel........................ 84 Colony PCR ................................................ 208, 249, 250 Copper(I)-catalyzed azide-alkyne conjugation (CuAAC)............................................... 65, 276 ascorbic acid and derived salts .................................. 41 Cu+ catalyst................................................................ 47 description ................................................................. 40 grafting, arginine-rich peptides ................................ 41 immobilizing peptides .............................................. 41

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onto chitosan............................................................. 41 optimization .............................................................. 47 peptide-chitosan conjugation ............................. 42–45 reactions..................................................................... 41 unreacted azide groups ............................................. 48 versatility .................................................................... 42 Covalent conjugation chemistry .................................... 56 CRISPR-Cas9....................................................... 191, 192 CRISPR-Cas9/λ-Red system ....................................... 208 Cyclic peptides aggregation.............................................................. 255 cell-based functional assays ..................................... 239 DNA sequencing..................................................... 256 E. coli .............................................................. 245–248 functional screening ................................................ 241 MisP ......................................................................... 256 protein misfolding................................................... 255 SICLOPPS............................................. 238, 239, 241 Cyclization..................................................................... 177 Cyclooctynes ................................................................... 65 Cysteine scanning strategy............................................ 180

D DARPins ........................................................................ 130 Deoxynucleotide (dNTP)............................................. 244 Deselenization ................................................................. 11 Desulfurization................................................... 31, 34, 35 Dhvar-5 peptide derivative .......................................43, 44 Dibenzocyclooctyne-maleimide (DBCO-maleimide) ...................................... 57 Difficult junctions ............................................................. 3 Dimer synthesis ............................................................... 78 Dimethylchlorosilane peptide......................................... 86 Diphosphine ligand....................................................... 218 Directed evolution ............................................... 189, 190 Diselenide catalysts......................................................4, 11 Diselenol catalyst............................................................... 4 DNA-based sequences .................................................. 184 DNA cloning ........................................................ 243, 244 DNA sequencing.................................................. 256, 261

E Ellman’s reagent........................................................55, 64 Enzymes......................................................................... 243 Epidermal growth factor receptor (EGFR) ................. 179 Error-prone PCR (ep-PCR) ......................................... 207 Escherichia coli (E. coli) cyclic peptides................................................. 245–248 MisP-GFP ....................................................... 240, 241 monitoring protein aggregation.................... 249, 253 monitoring protein misfolding...................... 249, 253 PMDs....................................................................... 240 pSICLOPPS library................................................. 246 strains ....................................................................... 243

PEPTIDE

AND

PROTEIN ENGINEERING: FROM CONCEPTS

Escherichia coli, chromosomal gene libraries CRISPR-Cas9 and λ-red technologies cassette assembly ...................................... 199, 200 donor DNA cassette design.............................. 198 E. coli cells harboring pCas...................... 200, 201 library construction........................................... 198 linear cassette assembly ............................ 198, 199 plasmid curing .......................................... 201, 202 principle ...................................192, 194, 196, 197 pTarget validation .................................... 193, 198 sgRNAs and cloning ......................................... 193 transformation ................................................... 201 verification, genome modification.................... 201 materials electroporation .................................................. 191 growth media .................................................... 191 strains and plasmids .......................................... 191 Extracellular matrix (ECM) cell signaling molecules............................................. 38 organization and roles .............................................. 38

F Fibronectin .................................................................... 129 First-generation library ................................................. 139 Fluorescence-activated cell sorting (FACS) ....................................... 241, 262, 263 Fluorinated pharmaceuticals........................................... 54 Fluorine atom.................................................................. 54 Fluoroglycoprotein, SPAAC AnxV with DBCO-bromoacetamide ......................60, 61 with DBCO-maleimide.................................57, 58 with Ellman’s reagent ......................................... 56 AnxV-S-acetamide-DBCO with FDGN3 ................................................. 63 AnxV-S-maleimide-DBCO with FDGN3 ............................................61, 62 LC–MS/MS .......................................... 55, 56, 58, 59 manipulations ............................................................ 56 protein mass spectrometry equipment..................... 55 reagents and solvents ................................................ 55 Fluoroglycoproteins ........................................................ 54 Free binding sites (FBS) .....................221, 223, 229, 233 Functionalization building block ........................................................... 69 glass ............................................................................ 88 hybrid bioorganic ordered mesoporous silica synthesis ......................................................... 82 hybrid peptides.......................................................... 70 OMS ....................................................................82, 87 silicone ....................................................................... 87 SiNPs ...................................................................89, 90 Si-O-metal covalent bonds ....................................... 69 titanium ...............................................................88, 89 with hybrid peptide................................................... 86

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G Genetic diversity............................................................ 190 Genotype–phenotype mapping .................................... 190 Gibson assembly................................................... 136, 277 Glass functionalization..............................................87, 88 Golden Gate–like approach .......................................... 138 Grafting peptides glass functionalization.........................................87, 88 silica grafting ............................................................. 87 titanium functionalization ..................................88, 89 Green chemistry approach.............................................. 47 Green fluorescent protein (GFP) ................................. 240 Guanidine hydrochloride................................................ 11

H HATU (N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b] pyridin-1-ylmethylene]-N methylmethanaminium hexafluorophosphate N-oxide)..................................... 16, 19, 21, 25 Hexahistidine-tagged target protein ................................271, 273, 275, 278 High-resolution mass spectroscopy (HRMS) ....................................................... 118 High-throughput screening ........................................253, 254, 257 Homogeneous glycoconjugates ..................................... 54 Homogeneous transition metal–catalyzed processes....................................................... 216 Homologous recombination .............................. 192, 195, 196, 202, 204, 208 Hoyveda-Grubbs 2nd generation catalyst .................... 218 Https traffic ..................................................................... 35 Huisgen’s 1,3-dipolar cycloaddition.............................. 40 Hybrid peptide-containing matrices hybrid hydrogels .................................................83, 84 peptide-silica hybrid matrices ................................... 82 Hybrid peptides silyl group alkoxysilanes ........................................................ 71 analyses ..........................................................72, 73 condensation ....................................................... 71 peptide silylation ................................................. 72 silylated reagents ................................................. 71 silylation carboxylic acid silane derivative .......................... 75 ICPTES ............................................................... 75 PEG...................................................................... 73 peptide ................................................................. 74 silyl group on peptides........................................ 75 on solid support ............................................77, 78 synthesis, peptide precursor.......................... 74–76 sol-gel process ..................................................... 70–71 surface functionalization........................................... 86 Hybrid silylated peptides ..........................................69, 90

PEPTIDE

286 Index

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PROTEIN ENGINEERING: FROM CONCEPTS

I Iminobiotin binding (IBB)........................................... 223 In vitro evolution ................................173, 176, 180, 181 In vitro selection ........................................................... 126 centyrins.......................................................... 179–180 double-stranded linear DNA.................................. 175 evolution of proteins...................................... 174–175 peptides........................................................... 176–177 Pin1 WW domain........................................... 178–179 VHH ............................................................... 177–178 In vitro selection strategy ............................................. 183 In vitro transcription translation (ITT) reaction......................................................... 182 Inducers of protein production.................................... 242 Inteins ............................................................................ 238 Interactive database, PCS-db ............................ 31, 32, 35 Intracellular fitness competition................................... 190 Islet amyloid polypeptide (IAPP)................................. 241

L Leucine-rich repeat proteins (LRR)............................. 130 Ligation ......................................................................... 260 Lipocalins....................................................................... 129 Luria-Bertani broth (LB).............................................. 242 Lyophilization ................................................................... 4 Lysis buffer .................................................................... 278

M Microbial hosts.............................................................. 239 Microscale thermophoresis (MST) .............................. 184 Midiprep plasmid kit..................................................... 248 Misfolded proteins (MisPs) .......................................... 238 MisP-GFP fusion.................................................. 255, 258 Monosilylated peptides ................................................... 81 Multiplexed automated genomic editing method ......................................................... 191

N Nano–differential scanning fluorimetry (nanoDSF) ................................................... 184 Nanomaterials ...........................................................93–96 Nanostructured bioorganic-inorganic materials self-assembly .............................................................. 85 templated assembly ................................................... 84 Native Chemical Ligation (NCL) ..................... 29, 32, 33 Natural transformation ................................................. 202 Network visualization software .................................... 245 Next-generation sequencing (NGS) ............................ 184 Non-canonical amino acids (ncAAs) AzK .......................................................................... 270 building blocks, protein engineering ..................... 267 incorporation efficiency .......................................... 273

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in-frame amber stop codon .................................... 268 orthogonal aaRS/tRNACUA pair ........................... 268 reactive side chains .................................................. 267 site-specific incorporation....................................... 268 Non-functionalized OMS nanoparticles........................ 87 Non-phagemid phage libraries ................... 135, 140, 142 Nε-[(2-azidoethoxy)-carbonyl]-L-lysine (AzK).......................................... 268, 270, 273

O Oligoethylene glycol (OEG), see Peptide-OEG conjugates Oligonucleotide ............................................................ 192 Open reading frame (ORF).......................................... 125 Overnight culture (ONC) ................................... 274, 278 Overnight induction ..................................................... 278

P pARCBD-p vector ............................................... 253, 261 pARCBDseqFor ............................................................ 261 PCR-free method .......................................................... 261 PCR reagents................................................................. 246 PCS website (pcs-db.fr) ............................................32, 33 Peptide silylation ............................................................. 72 Peptide-based materials ............................................38, 39 Peptide-modified silicone ............................................... 81 Peptide-OEG conjugates 4-benzyloxycarbonylmethyl-1,1,7,7-tetra (carboxymethyl)-1,4,7-triazaheptane trihydrochloride.................................. 102–104 bimodal platform 14 ...................................... 108–109 bimodal platform 15 ...................................... 109–111 cholesteryl tetraethylene glycol-cyclo(RGDfK) conjugate ............................................ 115–117 compounds 17–19 .................................................. 101 general laboratory equipment ............................97, 99 H-Arg-Gly-Asp-Ser-OEG-SH....................... 117–118 maleimide-OEG and thiol-containing peptide ........................................................... 95 monomodal platform 13 ........................................ 107 monovalent OEG-peptide platforms ..................... 101 multivalency.........................................................95, 96 multivalent platforms ........................................98–100 multivalent platforms 17–19 ......................... 114–115 nanomaterials ............................................................ 93 OEG derivatives ....................................... 96, 104–107 PEGylation ................................................................ 94 peptide moieties ............................................. 100–101 polymers .................................................................... 94 solvents and solutions .........................................97, 99 SPPS (see Solid-phase peptide synthesis (SPPS)) Peptides antimicrobial peptide ................................................ 43 biomedical research and drug development ............ 38

PEPTIDE

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PROTEIN ENGINEERING: FROM CONCEPTS

chemical synthesis ..................................................... 39 chemoselective peptide tethering ............................. 39 clinical and therapeutic applications......................... 39 C-terminally modified Dhvar-5 peptide derivative ........................................................ 44 CuAAC ...................................................................... 41 delivery routes ........................................................... 39 delivery systems carriers ............................................ 39 drug candidates ......................................................... 38 grafting ...................................................................... 41 peptide-chitosan conjugation .............................42, 43 sequences ................................................................... 38 synthesis, peptide-chitosan conjugates .................... 44 synthesis, peptide-chitosan ultrathin films............... 45 tethering ..............................................................42, 46 Peptidyl prolyl thioesters .................................................. 9 Phaberge helper phage ................................................. 145 Phage display affinity capture................................................ 142–143 alternative scaffolds ........................................ 128–131 antibody scaffold ............................................ 126–128 anticalins .................................................................. 129 artificial metalloenzymes......................................... 131 artificial repeat proteins .......................................... 130 artificially fused protein .......................................... 128 carboxy-terminus .................................................... 134 clones (see Screening individual clones) filamentous phages.................................................. 133 gene encoding ......................................................... 125 immunogenicity ...................................................... 128 level of display ......................................................... 142 libraries .......................................... 125, 127–130, 135 library of proteins.................................................... 138 measuring phage concentration .................... 141–142 oligonucleotide and gene synthesis methods ....................................................... 135 oligonucleotide-based libraries............................... 135 PEG (see Polyethylene glycol (PEG)) phage particles ................................................ 139–140 phage shock promoter (pPsp) ................................ 133 phagemid and phage libraries................................. 133 phagemid-based libraries ........................................ 135 phagemid-transformed bacteria ............................. 139 pLac ......................................................................... 130 protein binders .......................................125, 127–129 protein engineering................................................. 125 QuickLib......................................................... 136–137 replicative form (RF)............................................... 130 PhegemPhe pseudodipeptide sequence ......................... 85 Plasmid vectors.............................................................. 243 pMmOP plasmid ........................................................... 277 Polyethylene glycol (PEG) .......................................73, 94 CsCl equilibrium gradient ...................................... 141 dialysis ...................................................................... 141 precipitation.................................................... 140, 141

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Polymerase chain reaction (PCR) ................................ 183 Polymers amines into azides ...............................................44, 46 azide functional groups............................................. 41 biocompatible............................................................ 39 copolymers................................................................. 40 functionalization ....................................................... 38 Primers.................................................................. 192, 193 Protease inhibitors ........................................................ 229 Protein aggregation ...................................................... 237 Protein Chemical Synthesis Database (PCS-db) biological significance ............................................... 31 constraints.................................................................. 32 data management, in business intelligence.............. 31 description ................................................................. 31 interactive database ...................................... 31, 32, 35 language..................................................................... 35 PCS-GO module....................................................... 31 scenarios............................................................... 32–34 website ....................................................................... 31 Protein misfolding ........................................................ 237 Protein misfolding diseases (PMDs) aggregation.............................................................. 240 antibiotics ....................................................... 242, 243 Aβ42................................................................ 241, 242 combinatorial libraries ............................................ 241 common buffers ...................................................... 243 cyclic peptide libraries ............................................. 239 cyclic peptides.......................................................... 241 drug discovery process............................................ 241 E. coli ..................................................... 243, 249, 253 enzymes ................................................................... 243 equipment................................................................ 245 growth media ................................................. 242, 243 head-to-tail cyclic peptides, E. coli ................ 245–248 high-throughput screening .................. 253, 254, 257 inducer, protein production .......................... 242, 243 inteins....................................................................... 238 macrocyclic peptides ............................................... 238 microbial hosts ........................................................ 239 MisP ................................................................ 238, 240 MisP-GFP genetic system....................................... 240 peptide-based molecules ......................................... 238 plasmid vectors ........................................................ 243 primers ............................................................ 243, 244 protein folding ........................................................ 240 protein splicing............................................... 238, 239 proteinopathies/conformational diseases .............. 237 pSICLOPPS library................................246, 250–252 pSICLOPPS sub-library ................................ 254–256 reagents........................................................... 244, 245 SDS-PAGE/western blot reagents ........................ 244 SICLOPPS............................................. 238, 241, 249 SOD1.............................................................. 241, 242 sofware ..................................................................... 245

PEPTIDE

288 Index

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PROTEIN ENGINEERING: FROM CONCEPTS

Protein misfolding diseases (PMDs) (cont.) structure-activity relationships ...................... 257, 258 ultrahigh-throughput strategy................................ 242 Protein scaffolds ................................................... 174, 184 Protein splicing .................................................... 238, 239 ProteoFind script ............................................................ 35 pSICLOPPS library............................ 246–248, 250–252, 254, 260–262

Q QuickLib method.......................................................... 137

R Reactive side chains....................................................... 267 Ribosome display construct........................ 158, 159, 162 Ribosome display selection........................ 154, 157, 163, 164, 167, 168

S Sac7d-based randomized library .................................155, 157–159, 162 Sav-based artificial metalloenzymes 12 % acrylamide gel................................................. 223 5 % acrylamide gel ................................................... 225 biotinylated cofactor precursors ............................. 217 biotinylated ligand .................................................. 218 CFE and buffers ...................................................... 222 20 ZYP salts.................................................... 222 20 ZYP sugars ................................................ 222 200 mM MgSO4 ............................................... 222 ampicillin sodium salt ....................................... 222 antifoam solution .............................................. 222 auto-induction medium.................................... 222 LB agar plates .................................................... 222 LB medium........................................................ 222 LBSOB medium.................................................. 223 lysis buffer.......................................................... 223 RF1 buffer ......................................................... 223 RF2 buffer ......................................................... 223 detection, over-expressed T7Sav ............................ 228 elution buffer........................................................... 224 FBS determination .................................................. 229 FBS, in T7Sav tetramers ......................................... 223 IBB dialysis buffer ................................................... 223 large-scale overexpression, recombinant T7Sav .................................................. 226, 227 media and buffers DI-H2O/ultrapure water............................................................. 222 plasmid transformation, DE3 cells......................... 226 polyacrylamide gels and buffers ............................. 223 preparation, DE3 cells ................................... 225, 226 protein purification ........................................ 228, 229

TO

BIOTECHNOLOGICAL APPLICATIONS

protein staining solution......................................... 225 Screening individual clones clonal phage-ELISA ....................................... 143, 145 competitive phage-ELISA ...................................... 145 monoclonal phagemid particles ............................. 143 SDS-PAGE/western blot analysis .............. 250, 253, 255 SDS-PAGE/western blot reagents .............................. 244 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ................................................ 223 SEA ligation deionized water ........................................................... 4 deselenization ............................................................ 11 glove box ................................................................... 10 guanidine hydrochloride........................................... 11 HPLC analysis and purification.................................. 4 lyophilization ............................................................... 4 MALDI-TOF analysis ................................................. 5 MPAA ....................................................................2, 10 organic solvents and chemicals................................... 5 pH ............................................................................ 3, 4 at pH 4.0 catalyzed by diselenide 7b ..................... 8–9 at pH 5.5 catalyzed by MPAA................................ 6–7 principle ....................................................................... 2 SEA group ................................................................... 3 TCEP ........................................................................... 2 TCEP hydrochloride................................................. 10 TFA .............................................................................. 4 Selective protein modification ........................................ 54 Silica grafting................................................................... 87 Silicone grafting ...................................................................... 86 Silicone-based peptide polymers bis-silylated peptide................................................... 80 monosilylated peptides ............................................. 81 Silylated peptides.......................................................69, 86 Site-directed mutagenesis (SDM) ....................... 219, 226 Site-specific incorporation, ncAAs amber mutant, design and preparation.................. 273 AzK into target protein in E. coli .................. 274, 275 AzK-labeled target protein ............................ 275, 276 cloning, amber mutant ........................................... 274 CuAAC .................................................................... 276 orthogonal aaRS/tRNACUA pair ........................... 268 reagents and solutions ................................... 269–272 restriction cloning ................................................... 277 SpAAC ............................................................ 276, 277 specialized equipment............................................. 269 trace element stock solution .......................... 269, 271 translation stop codons........................................... 268 Sol-gel process advantage ................................................................... 70 bioorganic-inorganic structures ............................... 69 covalent networks...................................................... 71

PEPTIDE

AND

PROTEIN ENGINEERING: FROM CONCEPTS

hybrid hydrogels ....................................................... 83 hybrid peptides.................................................... 70–71 hydrolysis and condensation..................................... 70 hydroxyl groups ........................................................ 71 inorganic polymerization .......................................... 81 SiNPs ......................................................................... 89 Solid-phase peptide synthesis (SPPS) ............................................. 13–16, 100 cyclo(RGDfK) peptide................................... 113–114 Fmoc/tBu strategy ................................................. 110 general protocols............................................ 110–112 protected linear peptides ............................... 112–113 Stop codon suppression ................................................ 268 Strain-promoted azide-alkyne conjugation (SpAAC).............................................. 276, 277 Strain-promoted azide–alkyne cycloaddition (SPAAC)......................................................... 54 Streptavidin (Sav) bacterial origin......................................................... 219 biotin........................................................................ 214 catalysis .................................................................... 216 description ............................................................... 214 feature ...................................................................... 214 mature Sav ............................................................... 219 measurement, FBSs................................................. 229 overexpression, T7Sav ............................................ 220 permanent immobilization ..................................... 214 recombinant Sav gene ............................................. 219 T7-tag peptide......................................................... 219 temporary immobilization...................................... 214 tetrameric nature ..................................................... 216 toxic protein, E. coli ................................................ 229 Streptococcus thermophilus, chromosomal gene libraries materials growth media .................................................... 192 natural transformation ...................................... 192 natural competence chromosomal insertion, U-cat-oroP-D cassette ................................................ 203, 206 chromosomal insertion, U-lib-D library .................................................. 206, 207 exponential growth conditions......................... 202 natural transformation ...................................... 202 principle .....................................................202–204 U-cat-oroP-D cassette, assembling.................. 203 U-lib-D cassette, assembling ............................ 206 Sul7d proteins ............................................................... 158 SUMO-xN SEAoff peptide ................................. 23–25, 27 Super optimal broth (SOB).......................................... 242 Superoxide dismutase (SOD1)..................................... 241 Surface plasmon resonance (SPR)................................ 184

TO

BIOTECHNOLOGICAL APPLICATIONS Index 289

T T7-tagged mature streptavidin (T7Sav) ............ 219, 220, 225, 226, 228 Tailor-made materials...................................................... 37 TCEP hydrochloride....................................................... 10 Therapeutic molecules .................................................. 177 Thiol chemistry ............................................................... 54 Thiols ...........................................................................3, 10 Titanium functionalization .......................................88, 89 Transition metal catalysis .................................................................... 216 compatibility............................................................ 216 Trialkoxysilane derivatives .............................................. 81 Trialkoxysilylated peptides .............................................. 73 Trifluoroacetic acid (TFA) ............................... 4–9, 11, 16 Tris(2-carboxyethyl)phosphine (TCEP)..........................................2–4, 6, 8, 10 Tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl)..................................................... 5 Tumor necrosis factor alpha (TNF-α) ......................... 179

V Vascular endothelial growth factor receptor 2 (VEGFR2) ................................................... 178 VHH gene library ......................................................... 128

W Western blot analysis ..................................................... 251 Whole-bacterium ribosome display selection additional selection rounds..................................... 164 bacterial growth ...................................................... 157 clone screening and sequencing ............157, 166–167 ELISA ...................................................................... 157 follow-up of selection ............................................. 166 in vitro transcription ..............................156, 162–163 in vitro translation................................................... 156 ligation ..................................................................... 156 PCR................................................................. 155, 156 preparation, input library assembly, ribosome display construct .............. 162 pFP1001_tolA vector, generation........... 160, 161 randomization, sac7d gene...................... 159, 160 tolA fragment, feneration ................................. 161 primers ..................................................................... 155 pure recombinant protein....................................... 154 ribosome display selection, in bacterial suspension........................................... 163–165 selection strategies.......................................... 154, 156 strains ....................................................................... 154 Whole phage engineering ............................................. 125