Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols (Methods in Molecular Biology, 2676) 1071632507, 9781071632505

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Methods in Molecular Biology 2676

Yu-Hsuan Tsai · Simon J. Elsässer  Editors

Genetically Incorporated Non-Canonical Amino Acids Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Genetically Incorporated Non-Canonical Amino Acids Methods and Protocols

Edited by

Yu-Hsuan Tsai Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

Simon J. Els€asser Laboratory of Synthetic and Systems Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Solna, Stockholm, Sweden

Editors Yu-Hsuan Tsai Institute of Molecular Physiology Shenzhen Bay Laboratory Shenzhen, China

Simon J. Els€asser Laboratory of Synthetic and Systems Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology Karolinska Institutet Solna, Stockholm, Sweden

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3250-5 ISBN 978-1-0716-3251-2 (eBook) https://doi.org/10.1007/978-1-0716-3251-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 This work is subject to copyright. All rights are solely and exclusively licensed 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. This Humana 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 Noncanonical amino acids (ncAAs), or unnatural amino acids, can be utilized as protein building blocks beyond the natural repertoire dictated by the universal genetic code. ncAA can feature chemically diverse side chains which are readily incorporated into proteins by the ribosome. In principle, there are four routes for expanding the genetic code with ncAAs: first, exploiting natural substrate promiscuity of an endogenous aminoacyl-tRNA synthetase (aaRS) to charge its cognate tRNA with a ncAA similar to its natural substrate; second, introducing an engineered aaRS of an endogenous tRNA with expanded substrate specificity; third, introducing an orthogonal tRNA/aaRS pair to recode a sense codon; and fourth, introducing an orthogonal tRNA/aaRS pair to recode one of the stop codons. The fourth approach known as genetic code expansion (GCE), most commonly applied to amber stop codons to achieve ‘amber suppression,’ allows for site-specific incorporation of the desired ncAA. Together, the different strategies provide complementary means to expand the alphabet of available proteinogenic building blocks in bacteria, yeast, mammalian cells, and various model organisms. Many useful chemical groups have been introduced into proteins as side chains of ncAAs, including photolabile protection groups, biorthogonal handles, and fluorescence or spectroscopic probes. Thus, GCE has found a unique place in the chemical biologist’s toolbox, allowing researchers to probe and manipulate protein function in novel ways. Since their first conceptions, many GCE methods have matured into robust and well tested tools. They are ready to be exploited by cell and organismal biologists to answer research questions previously out of reach. Easy to follow, detailed, robust, and generalizable protocols are a crucial resource to facilitate the adoption of experts’ techniques as widely used research methodologies. This book aims to provide a broad resource of methods for implementing GCE in E. coli, mammalian cells, and animals, as well as to highlight specific applications ranging from fluorescence labeling to photocontrol and the study of protein post-translational modification. The first part focuses on GCE in E. coli: Chapter 1 describes a facile method to evolve synthetase variants with new substrate specificities. Chapters 2–5 describe applications of ncAAs for engineering proteins with new functionalities using amber suppression. Chapter 6 combines this strategy with biosynthesis of the ncAA in E. coli. Chapter 7 adds a strategy for recoding the initiator codon to encode multiple distinct ncAAs, and Chapter 8 implements ncAA mutagenesis in phage-display. Chapters 9 and 10 detail the preparation of recombinant proteins with post-translational modifications. The second part presents methods for mammalian cells: Chapters 11 and 12 describe general strategies to engineer mammalian cells for GCE, and Chapter 13 introduces an approach for quadruplet-codon suppression. Chapters 14–17 highlight the full breadth of ncAA application for studying function of proteins in mammalian cells, such as fluorescent labeling, photocontrol, and mimicking post-translational modification.

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Preface

The third part explores genetic code expansion in animals with Chapters 18 and 19 covering ncAA incorporation in zebrafish and mice, respectively. It is apparent that the strategies presented in the individual chapters can be adapted and extended, migrated to different model systems, and combined in new ways to help explore a wide range of biological questions and to augment industrial and pharmaceutical protein engineering. Shenzhen, China Solna, Sweden

Yu-Hsuan Tsai Simon J. Els€ a sser

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

PART I

GENETIC CODE EXPANSION IN E. COLI AND ITS APPLICATIONS

1 Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nikolaj G. Koch and Nediljko Budisa 2 Engineering Homogeneous Photoactive Antibody Fragments . . . . . . . . . . . . . . . . Thomas Bridge and Amit Sachdeva 3 Repurposing Photosensitizer Proteins Through Genetic Code Expansion to Facilitate Photo-Biocatalysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jiangyun Wang, Yan Xia, and Xuzhen Guo 4 Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities . . . . . . . . . . . . . . . Manjia Li and Tao Peng 5 Creating Selenocysteine-Specific Reporters Using Inteins . . . . . . . . . . . . . . . . . . . . Christina Z. Chung, Dieter So¨ll, and Natalie Krahn 6 Protein Expression with Biosynthesized Noncanonical Amino Acids. . . . . . . . . . . Yong Wang, Wenkang Cai, Boyang Han, and Tao Liu 7 Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli . . . . . . Han-Kai Jiang and Jeffery M. Tharp 8 Encoding Noncanonical Amino Acids into Phage Displayed Proteins. . . . . . . . . . Cristina Dı´az-Perlas, Montserrat Escobar-Rosales, Charles W. Morgan, and Benjamı´ Oller-Salvia 9 Genetically Encoded Noncanonical Amino Acids in Proteins to Investigate Lysine Benzoylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An-Di Guo and Xiao-Hua Chen 10 Semisynthesis of Glutamine-Methylated Proteins Enabled by Genetic Code Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Weimin Xuan and Xiaochen Yang

PART II 11 12

v ix

3 21

41

55 69 87

101 117

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147

APPLICATIONS IN MAMMALIAN CELLS

Genetic Code Expansion in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Zhigang Wu and Jie Wang Generation of Amber Suppression Cell Lines Using CRISPR-Cas9 . . . . . . . . . . . 169 Birthe Meineke and Simon J. Els€ a sser

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Genetic Code Expansion in Mammalian Cells Through Quadruplet Codon Decoding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yan Chen, Tianyu Gao, Xinyuan He, Wei Niu, and Jiantao Guo 14 Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation . . . . . . . . . . . . . . . . . . . Han Sun, Yang Huang, and Yu-Hsuan Tsai 15 Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Noncanonical Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maria Kowalski-Jahn, Hannes Schihada, and Gunnar Schulte 16 Selective Inhibition of Kinase Activity in Mammalian Cells by Bioorthogonal Ligand Tethering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jinghao Chen, Yang Huang, Wen-Biao Gan, and Yu-Hsuan Tsai 17 Site-Specific Incorporation of Sulfotyrosine into Proteins in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xinyuan He, Yan Chen, Jiantao Guo, and Wei Niu

PART III

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GENETIC CODE EXPANSION IN OTHER MODELS

18

Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code. . . . . . . . . . . . . . . . . . . . . . 247 Wes Brown, Carolyn Rosenblum, and Alexander Deiters 19 Noncanonical Amino Acid Incorporation in Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Zhetao Zheng and Qing Xia Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285

Contributors THOMAS BRIDGE • School of Chemistry, University of East Anglia, Norwich, UK WES BROWN • Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA a t Berlin, NEDILJKO BUDISA • Biocatalysis Group, Institute of Chemistry, Technische Universit€ Berlin, Germany; Chemical Synthetic Biology, Department of Chemistry, University of Manitoba, Winnipeg, MB, Canada WENKANG CAI • State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China JINGHAO CHEN • Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China; School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China XIAO-HUA CHEN • School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China; State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China YAN CHEN • Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA CHRISTINA Z. CHUNG • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA ALEXANDER DEITERS • Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA ` (IQS), Universitat Ramon Llull, CRISTINA DI´AZ-PERLAS • Institut Quı´mic de Sarria Barcelona, Spain SIMON J. ELSA€ SSER • Laboratory of Synthetic and Systems Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Solna, Stockholm, Sweden ` (IQS), Universitat Ramon MONTSERRAT ESCOBAR-ROSALES • Institut Quı´mic de Sarria Llull, Barcelona, Spain WEN-BIAO GAN • Institute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen, China TIANYU GAO • Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA AN-DI GUO • School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China; State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China JIANTAO GUO • Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA; The Nebraska Center for Integrated Biomolecular Communication (NCIBC), University of Nebraska-Lincoln, Lincoln, NE, USA XUZHEN GUO • CAS Key Laboratory of Quantitative Engineering Biology, Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

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Contributors

BOYANG HAN • State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China XINYUAN HE • Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA YANG HUANG • Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China; School of Basic Medical Sciences, Capital Medical University, Beijing, China HAN-KAI JIANG • Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Chemical Biology & Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan; Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan NIKOLAJ G. KOCH • Bioanalytics Group, Institute of Biotechnology, Technische Universit€ at Berlin, Berlin, Germany; Biocatalysis Group, Institute of Chemistry, Technische Universit€ at Berlin, Berlin, Germany MARIA KOWALSKI-JAHN • Receptor Biology & Signaling, Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden NATALIE KRAHN • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA MANJIA LI • State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China TAO LIU • State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China BIRTHE MEINEKE • Laboratory of Synthetic and Systems Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Solna, Stockholm, Sweden CHARLES W. MORGAN • Research School of Biology, The Australian National University, Canberra, ACT, Australia WEI NIU • Department of Chemical & Biomolecular Engineering, University of NebraskaLincoln, Lincoln, NE, USA; The Nebraska Center for Integrated Biomolecular Communication (NCIBC), University of Nebraska-Lincoln, Lincoln, NE, USA ` (IQS), Universitat Ramon Llull, BENJAMI´ OLLER-SALVIA • Institut Quı´mic de Sarria Barcelona, Spain TAO PENG • State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China; Institute of Chemical Biology, Shenzhen Bay Laboratory, Shenzhen, China CAROLYN ROSENBLUM • Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA AMIT SACHDEVA • School of Chemistry, University of East Anglia, Norwich, UK HANNES SCHIHADA • Receptor Biology & Signaling, Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden GUNNAR SCHULTE • Receptor Biology & Signaling, Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden DIETER SO¨LL • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA; Department of Chemistry, Yale University, New Haven, CT, USA HAN SUN • Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

Contributors

xi

JEFFERY M. THARP • Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA YU-HSUAN TSAI • Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China JIANGYUN WANG • Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; CAS Key Laboratory of Quantitative Engineering Biology, Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China; School of Future Technology, University of Chinese Academy of Sciences, Beijing, China JIE WANG • Department of Chemistry, Southern University of Science and Technology, Shenzhen, China YONG WANG • State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China ZHIGANG WU • Department of Chemistry, Southern University of Science and Technology, Shenzhen, China QING XIA • State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing, China YAN XIA • Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; School of Future Technology, University of Chinese Academy of Sciences, Beijing, China WEIMIN XUAN • School of Life Sciences, Tianjin University, Tianjin, China XIAOCHEN YANG • State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin, China ZHETAO ZHENG • State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing, China

Part I Genetic Code Expansion in E. coli and Its Applications

Chapter 1 Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids Nikolaj G. Koch and Nediljko Budisa Abstract The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals. Key words Pyrrolysyl-tRNA synthetase, Orthogonal translation, Stop-codon suppression, Protein engineering, Directed evolution, Synthetic biology

1

Introduction The expansion of the genetic code has proven to be an important tool to add new chemical structures to the biological world and to expand the chemical space of proteins beyond the 20 standard amino acids [1–3]. In this context, amino acids with non-proteinogenic functional groups (i.e., noncanonical amino acids, ncAAs) can be used to manipulate, design, and elucidate protein structure, dynamics, function, allosterism, interactions, catalysis, folding, synthesis, trafficking, degradation, and aggregation [4–10]. To facilitate this process, orthogonal translation systems (OTSs) have been developed. An OTS consists of an orthogonal pair (o-pair): an aminoacyl-tRNA synthetase (aaRS) and its cognate suppressor tRNA. The hallmark of OTSs is the reprogramming of protein translation with ncAAs, which requires (i) their metabolic compatibility and tolerance, (ii) translational

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Nikolaj G. Koch and Nediljko Budisa

Fig. 1 The tRNAPyl recognition mechanism of M. barkeri PylRS containing the N-terminal fused small metal binding protein (SmbP) domain [11, 12]. The 3D model structure (shown as cartoon) was calculated with ColabFold (N-terminal domains in light pink and C-terminal domain in blue) [13]. The model was created by aligning the N- and C-terminal domains with a superposition of the corresponding domains of M. mazei (PDB: 5UD5) and D. hafniense (PDB: 2ZNI) which are bound to the tRNAPyl [14, 15]. The conserved residues of the active site are shown as red sticks. For clarity and because the relative position is not clear, the linker region has been omitted. The N- and C-terminal domains of PylRS recognize the tRNAPyl in the anticodon stem region. The tRNAPyl, unlike canonical tRNAs, has only a tiny variable arm, which explains the orthogonality to all other canonical aaRS/tRNA pairs. It can also be seen that the anticodon is not involved in the recognition mechanism

orthogonality, and (iii) high fidelity of ribosomal synthesis in cells. Orthogonality in this context means that these aaRSs are capable of selectively recognizing their cognate tRNAs and ncAAs (Fig. 1) in the pool of endogenous tRNAs, aaRSs, metabolic amino acid intermediates (e.g., homoserine, homocysteine, citrulline, ornithine, etc.), and 20 canonical amino acids. Most of the previous OTSs derived from the archaea Methanosarcina mazei/barkeri PylRS (MmPylRS/MbPylRS) or Methanocaldococcus jannaschii tyrosyltRNA synthetase (MjTyrRS) which meet these criteria well [1–3]. PylRS is particularly powerful and its variants can incorporate various amino acid substrates into a protein. In fact, relatively simple engineering of PylRS allows us to incorporate almost all types of substrates (Fig. 2): long/heavy, charged, polar, small aliphatic, and aromatic including amino acids with “small-tag” side chains (e.g., S-allylcysteine, Sac). The original substrate of the PylRS/tRNAPyl pair is pyrrolysine (Pyl, 1), a lysine analog with a 4-methyl-pyrroline-5-carboxylate ring on the lysine side chain (Fig. 2). The anticodon of cognate tRNA is CUA, resulting in a natural orthogonal PylRS/tRNAPyl pair. Already in its natural cellular context in the genus Methanosarcina, this pair fulfills all the

Focused Engineering of Pyrrolysyl-tRNA Synthetase Discovery of PyIRS system

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Timeline 2002

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Fig. 2 Flowchart showing the timetable of characteristic ncAAs that can be incorporated with PylRS variants. We categorize the engineering of PylRS into three waves. The first wave was immediately after the discovery in the 2000s, where native or slightly mutated PylRS variants were shown to incorporate pyrrolysine derivatives with functional groups (e.g., alkynes, alkenes, and azides for bioorthogonal conjugation) into recombinant proteins [16]. The second wave was initiated after the remarkable discovery by Kavran et al. [17] that PylRS and PheRS are structurally very similar even though they have low sequence similarity. Indeed, the PylRS enzymes could be readily reengineered for recognizing Phe/Tyr/Trp analogs [16, 18]. The third wave (red) achieved recently includes ncAAs with small side chains that can be charged, polar, aliphatic, or have useful “small-tag” handles (e.g., olefin, alkyne, azide, diazirin, cyclopropane, etc.) [19, 20]

requirements as a natural OTS and is capable of suppressing the in-frame amber stop codon [16]. From a protein engineering perspective, an interesting structural feature of these class II aaRSs is that new substrate recognition evolved through changes in the side chains within the binding pocket rather than through changes involving the position of the protein backbone or secondary structure which are necessary in class I aaRS. Therefore, new ncAAs are (either evolutionarily or rationally) more easily encoded by a class II aaRS than class I aaRS. This plasticity is also reflected in the fact that very few mutations, usually only two to four, are required to encode a new substrate by PylRS [16]. Together with the lack of an anticodon recognition domain (Fig. 1), the PylRS OTS is probably the most accessible tool for experimental genetic code expansion to date. The recently improved PylRS variant of M. barkeri [22] has been shown to have a much wider range in substrate recognition than M. mazei, which may even facilitate the engineering for more diverse substrates. One problem in the standard workflow of finding enzyme mutants with desired properties is the library creation step. In a nonspecialized laboratory, it is almost impossible to do without

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bias. Extensive quality control measures are normally required to prevent library bias. While it has always been claimed that up to six positions can be randomized and screened (as limited by the transformation efficiency of E. coli), in reality this limit is lower because the assumption of a fairly equal distribution of all randomized constructs is unlikely to be achieved in normal laboratory settings [23]. For example, we were able to show that the construct found using the double-sieve approach capable of incorporating S-allylcysteine (Sac) was only the second best construct among four tested mutants [19]. Approaches were developed to create smart libraries (effectively reducing the number of possible DNA constructs while retaining the amino acid diversity). However, these approaches are not suitable for nonspecialized research groups or at least the time spent to get familiar with the methods has a very bad return on investment profile (time needed to set up a protocol to output) [24]. Our approach takes advantage of the enhanced enzyme promiscuity (in comparison to PylRSs derived from other organisms) of the M. barkeri PylRS, stabilized with the small metal-binding protein (SmbP) from Nitrosomonas europaea, while avoiding several problems normally associated with classical double-sieve selection methods [23]. Our method can be performed with the final plasmids used for target protein production with no need for special selection plasmids, therefore avoiding the tedious cloning steps normally required when libraries are transferred from negative to positive selection, to final plasmids. Since it is likely that stepwise improvement of enzymes can be achieved, the need for complicated library creation and, more importantly, implementation of a selection procedure can also be avoided [25]. The implementation of a suitable dual-screen selection scheme can easily take a few months and is therefore simply unsuitable for nonspecialized laboratories. When randomizing one positon, calculations using the Toplib tool showed that the probability of finding the best variant, screening 96 clones, is 95.3% and the probability of finding at least one of the two best variants is 99.8% (with a yield of 85%, representing the lower limit of primer purity, and therefore, it is assumed to be the lower yield limit of the DNA constructs generated) [26]. Here we present the necessary workflow (Fig. 3c) to develop a PylRS variant for new substrates that are structurally not too distinct from the initial starting substrate, or to incrementally improve the catalytic efficiency of known variants. For the development of the PylRS system to recognize substrates with divergent chemical structure, we recommend the dual-screen selection approach, but with a commercially obtained library to provide the required diversity and bias correction.

Focused Engineering of Pyrrolysyl-tRNA Synthetase

7

Fig. 3 3D structures of PylRS variants focusing on the binding pocket in complex with the substrate analogs, with the corresponding catalytically critical residues, and the screening workflow [19]. The availability of the high-resolution structural data served as a rough guide for the mutation approach. Since only structures of M. mazei are available, they were used in a homology model for M. barkeri. The numbering of residues in parentheses reflects the numbering of M. barkeri, while the others refer to M. mazei. (a) Wild-type MmPylRS (PDB ID: 2Q7H) [17] with bound Pyl-AMP. (b) Mutant MmOMeRS (PDB ID: 3QTC) [21] with bound O-methyltyrosine-AMP-PNP. (c) Workflow for the selection and characterization of novel PylRS mutants. The evaluation of the selected orthogonal pairs is based on sfGFP fluorescence screening. Only variants with significantly increased fluorescence intensity are selected and used for further analysis, as they are able to read through the amber stop codon with the ncAAs and generate a full-length sfGFP protein. Final variants are compared in performance with and without supplied ncAA

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Nikolaj G. Koch and Nediljko Budisa

2

Materials All buffers should be prepared using ultrapure water and biochemical grade chemicals. Buffers, antibiotic stocks, and chemicals should be sterile filtered using 0.22 μm syringe filters, bottle-top vacuum filtration systems (e.g., Steritop), or autoclaves. Media can be prepared with deionized water. Plasmid maps can be found in Fig. 4.

2.1

1. pET-28a_His6-SUMO-sfGFP(R2amber)-strep [27]: This screening plasmid is based on the widely used pET system containing a kanamycin resistance gene, an N-terminal His6tag, and a C-terminal strep-tag. The expressed sfGFP carries an amber stop codon (TAG) at position R2 and is controlled by an inducible T7 promoter (see Note 1 for sequence information).

Plasmids

2. pTECH_SmbP-MbPylRS [19, 22]: This plasmid contains the orthogonal translation system (see Note 1 for sequence information). The PylRS is under the control of a very strong constitutive lpp promoter. The tRNAPyl is under the control of the proK promoter. 2.2 Chemical Competent E. coli Cells and Transformation

A

1. E. coli Bl21(DE3) and TOP10 cells. 2. Plasmid isolation kit. 3. 75 mM CaCl2(aq).

MGSS SUMOopt sfGFP RBS T7 His tag

B

GGSH-linker Smbp RBS Ipp

Strep tag SA linker T7

lacl

pET-28a his-SUMO-sfGFP(R2amber)-strep 6,286 bp

f1

pTCEH_Smb-MbPyIRS 4,153 bp

CmR

MbPyIRS

Prok pyIT cat

prok

KanR pBR322

p15A

Fig. 4 Plasmids for screening (see Note 1 for sequence information). (a) The pET-28a_His6-SUMO-sfGFP (R2amber)-strep plasmid contains a stop codon at position R2. (b) The pTECH_SmbP-MbPylRS plasmid contains the PylRS synthetase as well as the tRNAPyl necessary for an orthogonal translation system. The functional parts are color coded as follows: origin of replication (light blue), Shine-Dalgarno sequence (RBS) (green), promoter (light green), terminator (red), tRNA (magenta), and protein coding sequence (yellow)

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4. Storage buffer: 75 mM CaCl2, 15% glycerol. 5. 50 mL centrifuge tubes. 6. LB medium: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, pH 7. 7. 25% glucose solution (see Note 2). 8. 50 mg/mL kanamycin stock solution. 9. 37 mg/mL chloramphenicol stock solution. 10. 9 cm-diameter petri dishes. 11. 1.5 mL microcentrifuge tubes. 12. 14 mL culture tubes. 13. Benchtop shaker. 14. Incubator. 15. Shaker. 16. 2-L flask. 17. Temperature adjustable centrifuge. 2.3 Gene Randomization

1. High-fidelity polymerase (e.g., NEB Q5). 2. Designed primers (see Note 3). 3. Restriction enzyme DpnI. 4. PCR tubes. 5. PCR purification kit. 6. Thermocycler. 7. Agarose gel electrophorese setup. 8. Spectrophotometer. 9. GelRed® dye (Biotinum).

2.4 Screening by Intact Cell Fluorescence Readout

1. Restriction enzyme BsaI. 2. T4 ligase. 3. Chemically competent E. coli Bl21(DE3) containing pET-28a_His6-SUMO-sfGFP(R2amber)-strep plasmid. 4. LB agar: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 1.5% (w/w) agar. 5. 14 cm-diameter petri dishes. 6. 100–200 mM ncAA solution (see Note 4). 7. ZYP-5052 auto-induction medium [28]: Put 928 mL of ZY (10 g/L tryptone and 5 g/L yeast extract) in a 1-L bottle. Add 50 mL of 20xP (1 M Na2HPO4∙7H2O, 1 M KH2PO4, 0.5 M (NH4)2SO4.), 20 mL of 50x5052 (25% glycerol, 2.5% glucose,

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10% α-lactose), 2 mL of 1 M MgSO4, and 0.2 mL of 1000× trace elements (e.g., Teknova, cat. no. T1001) or as described by Studier [28] . 8. Clear and black flat-bottom 96-well plates (see Note 5). 9. Toothpicks. 10. Multichannel pipette. 11. Adhesive gas-permeable foil for 96-well plates. 12. Adhesive aluminum foil for 96-well plates.

3

Methods Unless otherwise specified, all reaction steps should be carried out at room temperature. When working with bacteria, a sterile environment is essential. For the preparation of LB agar plates, we suggest a volume of 50 mL for each 14 cm-diameter plate and 15 mL for each 9 cm-diameter plate.

3.1 Production of Chemically Competent Cells

1. Pre-chill all buffers and 60 of 1.5 mL microcentrifuge tubes on ice. 2. Inoculate 300 mL LB media in a 2-L flask with the overnight E. coli culture (1:100). BL21(DE) cells are needed for the screening and possibly TOP10 cells for high-quality plasmid isolates (see Note 6). 3. Grow the cells at 37 °C and 200 rpm until an OD600 of 0.4–0.5, and then chill on ice. Important: All steps from here on are on ice. 4. Harvest the cells by centrifugation at 4 °C and 2500 × g for 10 min in 50 mL centrifuge tubes. 5. Discard the supernatant and resuspend the pellet in 50 mL ice-cold 75 mM CaCl2(aq) and incubate for 30 min on ice. 6. Harvest the cells by centrifugation at 4 °C and 4000 × g for 5 min. Discard the supernatant. 7. Resuspend all the pellets in 5 mL of 75 mM CaCl2(aq) and pool afterwards into one 50 mL centrifuge tube. Add 75 mM CaCl2 (aq) until 50 mL are reached. 8. Harvest the cells by centrifugation at 4 °C and 4000 × g for 5 min. Discard the supernatant. 9. Resuspend the pellet in 6 mL ice-cold storage buffer. 10. Aliquot 100 μL in the pre-chilled microcentrifuge tubes, snap freeze in liquid nitrogen, and store at -80 °C.

Focused Engineering of Pyrrolysyl-tRNA Synthetase

3.2 Focused MbPylRS Gene Randomization

11

The technique used here is nonoverlapping inverse PCR combined with site-saturation mutagenesis performed with mutagenic primers with NNK (N = A/T/G/C; K = T/G) that randomize the designated positions [29] (see Note 7). Design the primers according to good scientific practice and include BsaI overhangs upstream and downstream of the site to be randomized (see Note 3). 1. Use the PCR protocol of the manufacturer. Perform eight reactions in parallel with 25 elongation cycles (see Note 8). Scale to 20 μL reaction volume and 0.1 ng template DNA (pTECH_SmbP-MbPylRS). 2. Check for successful amplification with an 1% agarose gel, with 0.5 μL GelRed® for a 20 mL Gel. Use 0.5 μL of PCR product, 4.5 μL deionized water, and 1 μL 6× loading dye (6 μL total volume). 3. Transfer 0.5 μL DpnI to each PCR reaction tube and incubate for at least 2 h at 37 °C. 4. Pool the successful reactions and use a PCR purification kit for purification. 5. Determine concentration and store at -20 °C.

3.3 Digestion and Ligation of Randomized PylRS Gene Plasmid

1. Digest the PCR product according to the manufacturer’s instruction. 2. Purify the digested PCR product according to the manufacturer’s instruction and determine concentration. 3. Ligate 50 ng of DNA according to the manufacturer’s instruction and incubate for 1 h (see Note 9).

3.4 Transformation of Chemically Competent Cells

1. Pre-chill the required number of 1.5 mL microcentrifuge tubes on ice. 2. Use the aliquots prepared in subheading 3.1 and transfer 50 μL to the pre-chilled microcentrifuge tubes. 3. For the reporter plasmid, pET-28a_His6-SUMO-sfGFP (R2amber)-strep, transfer 0.2 μL of 100–200 ng/μL plasmid stock to the cells. For transformation of the randomized gene plasmid, pTECH_SmbP-MbPylRS, add 2.5 μL of the ligation product to E. coli cells already containing the reporter plasmid pET-28a_His6-SUMO-sfGFP(R2amber)-strep. Flick the tube three times to mix (see Note 10). 4. Cool on ice for 30 min. 5. Heat shock at 42 °C for 45 s. Chill on ice for 3 min. 6. Add 750 μL of LB to the microcentrifuge tubes and recover the cells for 1 h at 37 °C and 700 rpm in a benchtop shaker. 7. For transformations with the reporter plasmid, plate 80 μL onto a 9 cm-diameter LB ager plate containing 50 μg/mL

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kanamycin. For transformation with the randomized pTECH_SmbP-MbPylRS plasmid, plate everything onto a 14 cm-diameter LB agar plate containing 50 μg/mL kanamycin, 37 μg/mL chloramphenicol, and 1% glucose. 8. Incubate overnight at 37 °C. 3.5 Screening by Intact Cell Fluorescence Readout

1. Transfer 100 μL LB containing 50 μg/mL kanamycin, 37 μg/ mL chloramphenicol, and 1% glucose into each well of a clear 96-well plate. 2. Pick the colonies with a toothpick from the overnight plate into the clear 96-well plate containing the medium. 3. Seal the plate with a gas-permeable foil and incubate for 24 h at 37 °C and 300 rpm in a shaker (see Note 11). 4. The next day, prepare a black 96-well plate with 100 μL of ZYP-5052 supplied with 100 μg/mL kanamycin, 37 μg/ mL chloramphenicol, and the appropriate ncAA in each well (see Note 12). 5. Remove the gas-permeable foil from the overnight 96-well plate and use 1 μL to inoculate the black 96-well plate (see Note 13). 6. Seal the clear 96-well plate with aluminum foil and store at 4 ° C. 7. Seal the black plate with gas-permeable foil and incubate it for 24 h at 37 °C and 300 rpm in a shaker. 8. The next day, prepare a clear 96-well plate and add 50 μL of ZYP-5052 to each well. 9. Remove the gas-permeable foil from the overnight plate and measure the fluorescence using a plate reader, with excitation at 481 nm and emission at 511 nm. 10. For optical density measurements, transfer 50 μL of culture to the 50 μL of ZYP-5052 already present in each well and measure at 600 nm (see Note 13). 11. After data evaluation (see Fig. 5) and selection of potential hits (using your preferred software, such as Excel, Origin, QtiPlot, etc.), use the 96-well plate stored at 4 °C to inoculate the overnight culture. Wipe the aluminum foil with 70% ethanol, puncture the foil with a 10 μL pipette tip, and transfer 1 μL to a 14 mL culture tube containing 5 mL of LB medium with 37 μg/mL chloramphenicol. 12. The next day, isolate the plasmid DNA using the miniprep kit and send it for sequencing (see Note 14). 13. Evaluate the sequencing data and identify unique constructs using your preferred analysis tool.

Focused Engineering of Pyrrolysyl-tRNA Synthetase

13

Fig. 5 Example for screening of selected mutants [19]. Comparison of Sac incorporation efficiency for SmbPMbPylRS constructs (SmbP is omitted from the figure for clarity) (a) MbPylRS(C313W) and variants mutated at position W382 and (b) MbPylRS(W382S) with variants mutated at position C313. Fluorescence was measured for intact E. coli BL21(DE3) cells expressing the His6-SUMO-sfGFP(R2amber)-strep reporter protein. Data (incl. Standard deviation) represent the mean of three biological replicates 3.6 Comparison of Unique Constructs by Intact Cell Fluorescence Readout

1. Use the isolated plasmid DNA of the identified hits and transform them into E. coli cells already containing the reporter construct, such as pET-28a_His6-SUMO-sfGFP(R2amber)strep (see Subheading 3.4), and plate them on LB agar plates inoculated with 50 μg/mL kanamycin, 37 μg/mL chloramphenicol, and 1% glucose. 2. For overnight cultures, inoculate the cultures with three single colonies for each construct. Use a 14 mL culture tube containing 2 mL of LB with 50 μg/mL kanamycin, 37 μg/mL chloramphenicol, and 1% glucose. Incubate them for 24 h at 37 °C and 220 rpm.

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3. Prepare a black 96-well plate containing 100 μL of ZYP-5052 with 100 μg/mL kanamycin, 37 μg/mL chloramphenicol, and for each variant with and without corresponding ncAA (see Note 15). 4. Add 1 μL of the overnight cultures to each well. Seal the plate with a gas-permeable foil and incubate for 24 h at 37 °C and 300 rpm in a shaker. 5. Remove the gas-permeable foil from the overnight plate and measure fluorescence using a plate reader, with excitation at 481 nm and emission at 511 nm. 6. For optical density measurements, transfer 50 μL of culture to the 50 μL of ZYP-5052 already present in each well and measure at 600 nm (see Note 13). 7. Evaluate the data using your preferred analysis software (see Fig. 5 as an example and Note 16).

4

Notes 1. DNA sequences of the key elements: pTECH_SmbP-MbPylRS (plasmid map of the backbone available on Addgene, e.g., #99222): lpp promoter: 5’- AACCCAGCGTTCGATGCTTCTTTGAGCGAAC GATCAAAAATAAGTGCCTTCCCATCAAAAAAA TATTCTCAACATAAAAAACTTTGTGTAATACTTG TAACGC-3’. SmbP-(GGSH-linker)-MbPylRS; note that we always used the Y349F mutant by default, as it is known that this mutation generally enhances the aminoacylation efficiency [30]: 5′- ATGAGCGGTCATACCGCACATGTTGATGAAG CAGTTAAACATGCCGAAGAAGCAGTTGCACA CGGTAAAGAAGGCCATACCGATCAGCTGCT GGAACATGCAAAAGAAAGTCTGACCCATGCC AAAGCAGCCAGCGAAGCCGGTGGTAATACCC ATGTTGGTCATGGTATTAAACATCTGGAAGAT GCCATCAAACATGGTGAAGAGGGTCATGTT GGTGTTGCGACCAAACACGCACAAGAAGCAA TTGAACATCTGCGTGCAAGCGAACATAAAAG CCATGGCGGCTCTCATATGGACAAAAAACCG CTGGACGTTCTGATTAGCGCAATTGGTCTG TGGATGAGCCGTACCGGCACCCTGCATAAAA TCAAACATCATGAAGTTAGCCGCAGCAAAG TCTATATTGAAATGGCATGTGGTGATCATCT GGTGGTGAATAATAGCCGTAGCTGTCGTACC GCACGTGCATTTCGTCATCACAAATATCGT

Focused Engineering of Pyrrolysyl-tRNA Synthetase

15

AAAACCTGTAAACGTTGCCGTGTTAGCGACGAAGATATTAACAATTTTCTGACCCGT AGCACCGAAAGCAAAAATTCAGTTAAAGTT CGTGTTGTGAGCGCTCCGAAAGTT AAAAAAGCAATGCCGAAAAGCGTTAGTCGT GCACCGAAACCTCTGGAAAATAGCGTT AGCGCAAAAGCAAGCACCAATACCAGCCGT AGCGTTCCGAGTCCGGCAAAAAGCACCCCGAATAGCAGCGTTCCGGCAAGCGCACCGGCACCGAGCCTGACCCGTTCACAGCTGGAT CGTGTTGAAGCACTGCTGAGCCCTGAAGAT AAAATCAGCCTGAATATGGCAAAACCGTTT CGTGAACTGGAACCGGAACTGGTTACCCGT CGTAAAAATGATTTTCAGCGTCTGTAT ACCAACGATCGCGAAGATTATCTGGGT AAACTGGAACGTGATATTACCAAATTTTTCGT GGATCGCGGTTTTCTGGAAATCAAAAGCCCGATTCTGATTCCGGCAGAATATGTTGAACGT ATGGGCATTAATAACGATACCGAACTGAGCAAACAAATCTTCCGCGTTGATAAAAATCTGT GTCTGCGTCCGATGCTGGCACCGACCCTGT ATAACTATCTGCGCAAACTGGATCGTATTCT GCCTGGTCCGATTAAAATCTTTGAAGTTGGT CCGTGCTATCGCAAAGAAAGTGATGGTAAAGAACACCTGGAAGAGTTTACGATGGTT AACTTTTGTCAGATGGGTAGCGGTTGT ACCCGTGAAAATCTGGAAGCACTGATTAAAGAGTTTCTGGACTATCTGGAAATTGACTTT GAAATTGTTGGCGATAGCTGCATGGTTTTT GGTGATACCCTGGATATTATGCATGGTGAT CTGGAACTGAGTAGCGCAGTTGTTGGT CCGGTTAGCCTGGATCGCGAATGGGGTATT GATAAACCGTGGATTGGTGCAGGTTTTGGT CTGGAACGTCTGCTGAAAGTTAT GCACGGCTTTAAAAACATTAAACGT GCAAGCCGTTCCGAGAGCTATTACAATGGT ATTAGCACCAACCTGTAA-3’. proK promoter-tRNAPyl-proK terminator: 5’-GGCTAACTAAGCGGCCTGCTGACTTTCTCG CCGATCAAAAGGCATTTTGCTATTAAGGG ATTGACGAGGGCGTATCTGCGCAGTAAGATG CGCCCCGCATTGGAAACCTGATCATGTAG ATCGAATGGACTCTAAATCCGTTCAGCCGGG TTAGATTCCCGGGGTTTCCGCCAAATTCG AAAAGCCTGCTCAACGAGCAGGCTTTTTTG CAT-3′. pET-28a_His6-SUMO-sfGFP(R2amber)-strep:

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5’-ATGGGCAGCAGCCATCATCATCATCATCA CGGTTCTGACTCCGAAGTCAATCAAGAA GCTAAGCCAGAGGTCAAGCCAGAAGTCAA GCCTGAGACTCACATCAATTTAAA GGTGTCCGATGGATCTTCAGAGA TCTTCTTCAAGATCAAAAAGACCA CTCCTCTGCGTCGTCTGATGGAA GCGTTCGCTAAAAGACAGGGTAAGGAAA TGGACTCCTTAAGATTCTTGTACGACGGTA TTAGAATCCAAGCTGATCAGACCCCTGAAGA TTTGGACATGGAGGATAACGATATTATTGA GGCTCATCGCGAACAGATTGGTGGCATGTA GAAAGGCGAAGAGCTGTTCA CTGGTGTCGTCCCTATTCTGGTGGAACTGGA TGGTGATGTCAACGGTCATAA GTTTTCCGTGCGTGGCGAGGGTGAAGGTGA CGCAACTAATGGTAAACTGACGCTGAA GTTCATCTGTACTACTGGTAAACTGCCGGTA CCTTGGCCGACTCTGGTAACGACGCTGA CTTATGGTGTTCAGTGCTTTGCTCGTTA TCCGGACCATATGAAGCAGCATGA CTTCTTCAAGTCCGCCATGCCGGAAGGCTA TGTGCAGGAACGCACGATTTCCTTTAAGGA TGACGGCACGTACAAAACGCGTGCGGAA GTGAAATTTGAAGGCGATACCCTGGTAAA CCGCATTGAGCTGAAAGGCATTGACTTTAAA GAAGACGGCAATATCCTGGGCCATAA GCTGGAATACAATTTTAACAGCCACAA TGTTTACATCACCGCCGATAAACAAAAAAA TGGCATTAAAGCGAATTTTAAAATTCGCCA CAACGTGGAGGATGGCAGCGTGCA GCTGGCTGATCACTACCAGCAAAACA CTCCAATCGGTGA TGGTCCTGTTCTGCTGCCAGACAATCACTA TCTGAGCACGCAAAGCGTTCTGTCTAAAGA TCCGAACGAGAAACGCGATCATA TGGTTCTGCTGGAGTTCGTAACCGCA GCGGGCATCACGCATGGTATGGATGAA CTGTACAAAAGCGCTTGGAGCCACCCGCA GTTCGAAAAATAA-3′. T7 promoter: TAATACGACTCACTATAGG. T7 terminator: CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTT GAGGGGTTTTTTG

Focused Engineering of Pyrrolysyl-tRNA Synthetase

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2. Should be sterile filtered and not autoclaved. 3. Use HPLC purified primers to increase the percentage of correctly amplified product, minimizing false-negative results during screening (see Introduction for more information). 4. ncAA stock solutions can be prepared in water or, depending on the ncAA stability and hydrophobicity, in 0.1–1 M NaOH. Since the medium is strongly buffered, this does not pose a problem for screening. In our laboratory, ncAAs dissolved in 1 M NaOH with a final concentration in medium of up to 10 mM can be screened without problems [19]. 5. Even though the combination of clear and black 96-well plates is the cheapest option, the black 96-well plate can be replaced by a black plate with a clear bottom to save the pipetting step for optical density measurements. The medium volume must be adjusted accordingly. 6. Important: Kanamycin must be added when preparing cells with the reporter plasmid (pET-28a_His6-SUMO-sfGFP (R2amber)-strep), as well as for overnight culture (see Subheading 3.3). 7. When mutating MbPylRS for small substrate recognition, residue at position N311 (M. barkeri notation) must always be as big as Val or bigger to ensure orthogonality [19]. If the enzyme is engineered for the recognition of Lys derivatives, N311 must be kept with C313 mutated to Val to ensure orthogonality [31]. 8. Using more but smaller reaction volumes and fewer cycling steps reduces amplification bias which is introduced due to the path-dependent nature of the amplification process. Meaning, run several small reactions in parallel. We usually run 8 × 20 μL reactions, which are then combined for purification. The bias almost certainly occurs when using NNK primers. This is because these primers do not have a perfect homogeneous distribution of their NNK codons. This means that the template that is amplified first becomes the dominant product, one of the main problems in library construction via PCR. 9. The reaction can be stored at 4 °C for at least a week and reused if necessary. 10. Do not pipette up and down to mix, as the cells in the transformation buffer are fragile. 11. This requires adhesive mats for normal flask shaker trays; otherwise, 96-well plate incubators must be used. 12. A higher concentration of kanamycin is necessary because efficacy is reduced in strongly buffered medium. Depending on what the target of engineering is, concentrations of 1–10 mM

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ncAA are feasible. Higher concentrations are recommended to find variants for more divergent substrates. 13. An 8- or 12-channel pipette is recommended. 14. If sequencing fails, use the same isolated plasmid DNA for transformation (see Subheading 3.4) of an E. coli cloning strain (e.g., TOP10) for subsequent isolation. 15. For each culture, one well should contain the ncAA and one without. In a 96-well plate, 16 constructs or 16 different ncAA concentrations can be tested in this way. 16. Now that triplicate experiments are performed and the background suppression data are available, a robust evaluation/ assessment (including error bars) of catalytic efficiency is possible. Furthermore, this step can be repeated, and the constructs can be compared as a function of ncAA concentration to gain further insight into catalytic efficiency. References 1. Mukai T, Lajoie MJ, Englert M, So¨ll D (2017) Rewriting the genetic code. Annu Rev Microbiol 71:557–577. https://doi.org/10.1146/ annurev-micro-090816-093247 2. Chin JW (2017) Expanding and reprogramming the genetic code. Nature 550:53–60. https://doi.org/10.1038/nature24031 3. Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444. https://doi.org/10. 1146/annurev.biochem.052308.105824 4. Groff D, Thielges MC, Cellitti S, Schultz PG, Romesberg FE (2009) Efforts toward the direct experimental characterization of enzyme microenvironments: tyrosine 100 in dihydrofolate reductase. Angew Chem Int Ed 48: 3478–3481. https://doi.org/10.1002/anie. 200806239 5. Baumann T, Hauf M, Schildhauer F, Eberl KB, Durkin PM, Deniz E, Lo¨ffler JG, AcevedoRocha CG, Jaric J, Martins BM, Dobbek H, Bredenbeck J, Budisa N (2019) Site-resolved observation of vibrational energy transfer using a genetically encoded ultrafast heater. Angew Chem Int Ed 58:2899–2903. https://doi. org/10.1002/anie.201812995 6. Minnihan EC, Young DD, Schultz PG, Stubbe J (2011) Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase. J Am Chem Soc 133:15942–15945. https://doi. org/10.1021/ja207719f 7. Li JC, Nastertorabi F, Xuan W, Han GW, Stevens RC, Schultz PG (2019) A single reactive

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fused solubility tags. Front Bioeng Biotechnol 9:1–14. https://doi.org/10.3389/fbioe. 2021.807438 23. Schmidt MJ, Summerer D (2018) Directed evolution of orthogonal pyrrolysyl-tRNA synthetases in Escherichia coli for the genetic encoding of noncanonical amino acids. In: Lemke E (ed) Noncanonical amino acids. Methods in molecular biology, vol 1728. Humana Press, New York, pp 97–111 24. Lacey VK, Louie GV, Noel JP, Wang L (2013) Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings. Chembiochem 14:2100–2105. https:// doi.org/10.1002/cbic.201300400 25. Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2: 891–903. https://doi.org/10.1038/nprot. 2007.72 26. Nov Y (2012) When second best is good enough: another probabilistic look at saturation mutagenesis. Appl Environ Microbiol 78: 258–262. https://doi.org/10.1128/AEM. 06265-11 27. Hauf M, Richter F, Schneider T, Faidt T, Martins BM, Baumann T, Durkin P, Dobbek H, Jacobs K, Mo¨glich A, Budisa N (2017) Photoactivatable mussel-based underwater adhesive proteins by an expanded genetic code. Chembiochem 18:1819–1823. https://doi.org/10. 1002/cbic.201700327 28. Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234. https://doi.org/10.1016/j.pep.2005.01.016 29. Dominy CN, Andrews DW (2003) Sitedirected mutagenesis by inverse PCR. In: Casali N, Preston A (eds) E. coli plasmid vectors. Methods in molecular biology, vol 235. Humana Press, Totowa, pp 209–223 30. Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S (2008) Multistep engineering of pyrrolysyltRNA synthetase to genetically encode Nε-(o-Azidobenzyloxycarbonyl) lysine for sitespecific protein modification. Chem Biol 15: 1187–1197. https://doi.org/10.1016/j. chembiol.2008.10.004 31. Hohl A, Karan R, Akal A, Renn D, Liu X, Ghorpade S, Groll M, Rueping M, Eppinger J (2019) Engineering a polyspecific pyrrolysyltRNA synthetase by a high throughput FACS screen. Sci Rep 9:11971. https://doi.org/10. 1038/s41598-019-48357-0

Chapter 2 Engineering Homogeneous Photoactive Antibody Fragments Thomas Bridge and Amit Sachdeva Abstract Genetically encoded site-specifically incorporated noncanonical amino acids (ncAAs) have been used to modulate properties of several proteins. Here, we describe a procedure for engineering photoactive antibody fragments that bind to their target antigen only after irradiation with 365 nm light. The procedure starts with identification of tyrosine residues in antibody fragments that are important for antibody–antigen binding and thus targets for replacement with photocaged tyrosine (pcY). This is followed by cloning of plasmids and expression of pcY-containing antibody fragments in E. coli. Finally, we describe a cost-effective and biologically-relevant method for measuring the binding affinity of photoactive antibody fragments to antigens expressed on the surface of live cancer cells. Key words Antibodies, Cancer, Photocaged amino acids, Noncanonical amino acids, Synthetic biology, Light-activated biotherapeutics, Genetic code expansion

1

Introduction The last two decades have seen a rapid increase in the use of monoclonal antibodies and antibody fragments in a variety of applications in medicine, biotechnology, and nanotechnology [1– 7]. These applications rely on the ability of the antibody to bind to its target antigen with high specificity and selectivity. Controlling the binding of antibody to its corresponding antigen in a userdefined manner can further expand the scope for these antibodies. Towards this goal, we and others have developed photoactive antibodies that bind to their target antigen upon irradiation with a specific wavelength of light [8–11]. Such photoactive antibodies could find applications in biotechnology by providing lightmediated spatial and temporal control over antibody-dependent processes, in biotherapeutics by controlling the binding of antibody-drug conjugates to cancer cells in a light-dependent manner, and in nanotechnology by providing light-activated antibodybased molecular switches.

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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One of the earliest methods for developing light-activated antibodies relied on nonspecific labelling of antibodies with 1-(2-nitrophenyl)ethanol to generate an inactive antibody [11]. Irradiation of these antibodies with ultraviolet-A radiation removed the 2-nitrophenyl caging group, thus liberating an active antibody. However, this approach relies on the use of inhomogeneous mixture of caged antibody, which is not ideal for applications in medicine and biotechnology. The expansion of the available crystal structures of antibody–antigen complexes has provided detailed structural understanding of the residues in the antibody that are important for mediating antibody–antigen binding. Using this information and methods to site-specifically incorporate noncanonical photocaged amino acids can allow development of photoactive antibodies for several target antigens. Genetic site-specific incorporation of noncanonical amino acids (ncAAs) into proteins expressed in live cells is achieved by assigning a rarely used amber stop codon or quadruplet codons to the ncAA and supplying the cells with an aminoacyl-tRNA synthetase (aaRS)/tRNA pair that is orthogonal (i.e., does not cross-react) to the host cell aaRS/tRNA pairs. This orthogonal aaRS specifically charges the orthogonal tRNA with the ncAA. The orthogonal tRNA has an anticodon that forms Watson–Crick base pairs with amber stop codon or quadruplet codon on the mRNA and is used for site-specific incorporation of ncAA in response to amber stop codon or quadruplet codons. Using this approach, close to 200 ncAAs have been genetically encoded in different organisms [12–15]. These include ncAAs containing functional groups for bioorthogonal conjugation, post-translational modifications, photoreactive functional groups, (photo)caged amino acids, and reactive amino acids. Here, we describe the use of site-specifically incorporated photocaged ncAAs, more specifically, photocaged tyrosine (pcY) to generate photoactive antibody fragments. In recent years, small antibody fragments such as scFv, Fab, and nanobodies have been developed for a variety of different targets [16–18]. These antibody fragments are attractive alternatives to full-length antibodies due to their small size, ease of genetic alteration, and synthesis in bacterial cells [19]. In this chapter, we provide detailed experimental procedure for (1) expression of ncAA-containing antibody fragments in E. coli and (2) assessing the binding of photocaged antibodies to target antigens expressed on the surface of cancer cells.

2

Materials

2.1 Cell Lines and Strains

1. Human epithelial carcinoma cell line A-431. 2. Human breast adenocarcinoma cell line MDA-MB231.

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3. E. coli BL21(DE3) pLysS strain. 4. E. coli DH10B strain. 5. E. coli XL10-Gold strain. 2.2

Equipment

1. Digital heating shaking dry bath. 2. Incubator. 3. Shaking incubator. 4. Horizontal gel electrophoresis apparatus. 5. PAGE gel apparatus. 6. Centrifuge. 7. Gel imager. 8. Microvolume spectrophotometer. 9. Disposable gravity-flow chromatography columns. 10. Vivaspin 500 MWCO 3 kDa. 11. Mass spectrometer. 12. CO2 cell culture incubator. 13. Microscope. 14. Hemacytometer. 15. TC-treated 96-well white plate. 16. 96-well clear plate. 17. Syringe filters with 0.2 μm pore PTFE membrane. 18. Sterile disposable filters with 0.2 μm PES membrane. 19. Multichannel pipette 30–300 μL. 20. Single-channel pipette 0.2–2.5 μL, 2–20 μL, 20–100 μL, and 100–1000 μL.

2.3

Software

1. UCSF Chimera. 2. SnapGene Viewer. 3. QuickChange Primer Design tool (https://www.agilent.com/ store/primerDesignProgram.jsp). 4. Codon Optimization Tool (https://sg.idtdna.com/pages/ tools/codon-optimization-tool). 5. ImageLab.

2.4

Reagents

1. Dulbecco’s modification of Eagle medium (DMEM). 2. Fetal bovine serum (FBS). 3. Penicillin and streptomycin for cell culture (PEN/STREP). 4. NEBuilder HiFi DNA Assembly (New England Biolabs). 5. Gene fragment.

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6. Restriction enzymes: DpnI, NdeI, NotI, and HindIII. 7. Plasmids: pSANG10-3F-BG4 (Addgene plasmid # 55756) and pULTRA-CNF (Addgene plasmid # 48215). 8. Kanamycin. 9. Miniprep kit. 10. Gel extraction kit. 11. Custom DNA oligos. 12. Site-directed mutagenesis kit. 13. Agarose powder. 14. 1-kb DNA ladder. 15. Spectinomycin. 16. Deionized water. 17. Glucose. 18. LB–agar powder. 19. LB–broth Miller powder. 20. Tryptone. 21. Yeast extract. 22. Isopropylthio-β-galactoside (IPTG). 23. Sucrose. 24. Tris–HCl. 25. EDTA. 26. MgCl2. 27. Ni-NTA agarose. 28. NaCl. 29. Imidazole. 30. HCl. 31. NaOH. 32. 4
LDS loading buffer. 33. 1,4-Dithiothreitol (DTT). 34. Phosphate-buffered saline (PBS) powder. 35. Dulbecco’s phosphate-buffered saline (DPBS). 36. 4–12% Bis–Tris gel 37. Protein standard for SDS-PAGE. 38. 10
MES SDS running buffer 39. Coomassie protein stain. 40. Nonfat dry milk powder. 41. 37% (w/v) formaldehyde.

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42. Antibodies: 6
-His tag monoclonal antibody (Invitrogen, #MA1-21315), anti-mouse IgG, HRP-linked antibody (Cell Signaling, #7076S). 43. Trypsin–EDTA (0.05%), phenol red. 44. Chemiluminescent substrate. 2.5 Solutions and Buffers

1. Complete medium: Add 56 mL FBS and 5.6 mL 100
PEN/STREP to 500 mL DMEM. 2. LB–agar: Dissolve 20 g LB–agar powder in 450 mL deionized water; adjust volume to 500 mL with deionized water. Sterilize by autoclaving and store at room temperature. To melt agar, microwave on the lowest setting or submerge agar bottle in water bath set to 50  C. 3. LB–broth: Dissolve 25 g LB–broth Miller powder in 900 mL deionized water; adjust volume to 1000 mL with deionized water. Sterilize media by autoclaving and allow to cool to room temperature before use. 4. 40% (w/v) glucose solution: Dissolve 400 g glucose powder in 500 mL deionized water using a heated stirrer plate. Once fully dissolved, adjust volume to 1000 mL using deionized water and autoclave to sterilize solution. 5. 2xTY: Dissolve 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 900 mL deionized water. Adjust volume to 1000 mL with deionized water. Sterilize media by autoclaving and allow to cool to room temperature before use. 6. 2xTY-GKS media: 2xTY media with 2% glucose, 50 μg/mL kanamycin, and 100 μg/mL spectinomycin. 7. 2xTY-GK1/2S1/2 media: 2xTY media with 2% glucose, 25 μg/ mL kanamycin, and 50 μg/mL spectinomycin. 8. Photocaged tyrosine (pcY) stock solution: 200 mM pcY in 1 M NaOH. 9. Periplasmic extraction buffer 1 (20% sucrose, 100 mM Tris– HCl, 1 mM EDTA, pH 8.0): Dissolve 200 g sucrose in 600 mL deionized water, and then add 100 mL 1 M Tris–HCl. Adjust pH to 8, and then add 4 mL 250 mM EDTA. Adjust volume to 1000 mL and then filter sterilize through a 0.2 μm filter unit. This can be stored at 4  C for several months. 10. Periplasmic extraction buffer 2 (5 mM MgCl2): Add 5 mL 1 M MgCl2 to 995 mL deionized water and then filter sterilize through a 0.2 μm filter unit. This can be stored at 4  C for several months. 11. 1x PBS buffer (137 mM NaCl, 2.7 mM KCl, 11.9 mM phosphates, pH 7.4): Dissolve 9.93 g phosphate-buffered saline powder in 1000 mL deionized water. Sterilize by autoclaving.

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12. Ni-NTA wash buffer (50 mM Tris–HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0): Dissolve 875 mg NaCl and 68 mg imidazole into 40 mL deionized water. Add 2.5 mL 1 M Tris–HCl and adjust pH to 8 followed by volume to 50 mL with deionized water. Filter through a 0.2 μm syringe filter and store at 4  C for up to a month. 13. Ni-NTA elution buffer (50 mM Tris–HCl, 300 mM NaCl, 200 mM imidazole, pH 8.0): Dissolve 875 mg NaCl and 680 mg imidazole into 40 mL deionized water. Add 2.5 mL 1 M Tris–HCl and adjust pH to 8 followed by volume to 50 mL with deionized water. Filter through a 0.2 μm syringe filter and store at 4  C for up to a month. 14. Fixing solution (3.7% formaldehyde): Add 1.5 mL 37% formaldehyde to 13.5 mL sterile deionized water. 15. Assay wash buffer, PBST (1
PBS, 0.1% Tween-20): Add 1 mL Tween-20 into 1 L PBS. 16. Blocking solution (10% milk, PBST): Dissolve 1 g milk powder in 10 mL PBST. 17. Primary antibody solution (1:1000, 1% milk, PBST): Add 0.5 mL blocking solution to 4.5 mL PBST followed by 5 μL of primary antibody. 18. Secondary antibody solution (1:1500, 1% milk, PBST): Add 0.5 mL blocking solution to 4.5 mL PBST followed by 3.33 μL of secondary antibody.

3

Methods

3.1 Identification of Candidate Tyrosine Residues in the Antigen Binding Site of an Antibody Fragment for Replacement with Photocaged Tyrosine

1. Select a crystal structure of antibody fragment bound to its antigen. If the crystal structure of the antibody–antigen complex is not available, use computational methods such as AlphaFold to predict the structure [20]. 2. Load the PDB file into the protein structure visualization software (e.g., UCSF Chimera). 3. Next, identify tyrosine residues in the antibody fragment that might be important for antibody–antigen binding. For this, use the zoning tool in UCSF Chimera (or equivalent in another software), and select tyrosine residues within 5.0 A˚ of the antigen. 4. These tyrosine residues could be selected for mutagenesis to photocaged tyrosine (pcY). If no tyrosine residue in the antibody is present at the antibody–antigen binding interface, then other residues could be selected (see Note 1).

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3.2 Expression of Photocaged TyrosineContaining Antibody Fragments in E. coli

Site-specific incorporation of a ncAA into proteins produced by live cells requires three essential components: (1) an expression plasmid containing the gene of interest with amber stop codon at the position assigned to ncAA, (2) a suppressor plasmid containing genes for the orthogonal aaRS/tRNACUA pair that are specific for ncAA, and (3) the ncAA. The origin of replication of the expression plasmid and the suppresser plasmid should be compatible with each other, so that they can coexist in the same cell. Also, these plasmids should have different antibiotic resistance genes. The expression plasmid used in our methods is pSANG10, which has been previously developed for efficient expression of antibody fragments [21, 22]. This plasmid allows expression and transport of the antibody fragments to the periplasm of E. coli that has an oxidizing microenvironment suitable for maintaining disulfide bonds in the protein. In our methods, this transport of the antibody fragment to the periplasm is mediated by the N-terminal PelB peptide. The peptide is removed during the transport to the periplasm. Also, our methods describe purification of antibody fragments using the C-terminal poly-histidine (His6) tag. In principle, this could be replaced with other purification tags at the C-terminus of the antibody fragment. The suppresser plasmid used in our methods is pULTRA that was developed for efficient site-specific incorporation of ncAAs in proteins expressed in E. coli [23]. These plasmids are available through Addgene. In the following subsections, we describe methods for (1) cloning of an antibody fragment into a pSANG10 plasmid, (2) cloning of pcY-specific evolved mutant of Methanocaldococcus jannaschii tyrosyl–tRNA synthetase (MjRS) into pULTRA suppressor plasmid, and (3) expression and purification of pcY-containing antibody fragments.

3.2.1 Cloning of Antibody Fragment and Its Amber Mutants into pSANG10 Plasmid

1. Using the amino acid sequence of the antibody fragment, design a codon-optimized gene fragment. Add a sequence corresponding to PelB at the 50 -end (Fig. 1). 2. Change the codon corresponding to the photocaged tyrosine to the amber codon TAG. 3. Add the 25 base pair overhangs at the 50 - and 30 -ends of the designed gene fragment, and order this as a duplex DNA from a custom gene synthesis supplier (Fig. 1). We name this gene fragment as “antibody gene fragment” (see Note 2). 4. Digest the pSANG10-3F-BG4 plasmid with NdeI and HindIII. In a 30 μL restriction digest reaction, add 1000 ng of pSANG10-3F-BG4 plasmid, 0.5 μL of 20,000 units/mL NdeI, and 0.5 μL of 20,000 units/mL HindIII. Incubate at 37  C for 1 h. Run the reaction mixture on 1% agarose gel and cut the band corresponding to the pSANG10-3F plasmid backbone (5.2 kb). Extract the pSANG10-3F plasmid backbone using a gel extraction kit.

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Fig. 1 Cloning of expression and suppressor plasmids employed for site-specific incorporation of photocaged tyrosine (pcY) into antibody fragments expressed in E. coli. (a) Antibody gene fragment designed for insertion into pSANG10 plasmid. The gene fragment had 25 base pair overhangs at the 50 - and 30 -ends to allow cloning using NEBuilder/Gibson cloning. PelB leader peptide mediates transport of the antibody fragment to the periplasm of E. coli. pSANG10-3F-BG4 is digested with NdeI and HindIII restriction endonucleases, and the antibody gene fragment is inserted using NEBuilder/Gibson cloning to generate pSANG10-antibody fragment plasmid. (b) Gene fragment for pcY-specific mutant of Methanocaldococcus jannaschii tyrosyl–tRNA synthetase (MjpcYRS). The gene fragment had 25 base pair overhangs at the 50 - and 30 -ends to allow cloning using NEBuilder/Gibson cloning. pULTRA-CNF plasmid is digested with NotI restriction endonuclease, and MjpcYRS gene fragment is inserted using NEBuilder/Gibson cloning to generate pULTRA-pcY plasmid

5. Next, clone the antibody gene fragment into the digested pSANG10 plasmid backbone using NEBuilder or a similar method. In a 10 μL reaction, add 5 μL of 2
NEBuilder (or Gibson cloning) Master Mix, 100 ng of digested pSANG10-3F plasmid backbone, and twofold molar excess of the antibody gene fragment, and adjust volume to 10 μL with Ultrapure water (see Note 3). 6. Incubate the reaction for 1 h at 50  C. 7. Transform the reaction mixture into DH10B cells. For this, add 2 μL of the reaction mixture to 50 μL DH10B chemically competent cells. Incubate on ice for 15 min, heat shock by incubating at 42 C for 45 s, and transfer to ice. Incubate on ice for another 5 min and add 500 μL SOB media. Recover the cells by incubating this mixture at 37 C for 1 h, with shaking on a thermomixer at 900 rpm. 8. After 1 h, pellet the cells by centrifugation at 10,000 g and 4  C for 1 min. Discard 400 μL of the supernatant and resuspend the cells with the rest of the media. 9. Plate these cells on LB–agar plates supplemented with 50 μg/ mL kanamycin and incubate overnight at 37  C. 10. Select 3–5 colonies and grow overnight in LB–broth (37  C, 200 rpm) supplemented with 50 μg/mL kanamycin. Extract plasmid DNA using a Miniprep kit and confirm the correct insertion using Sanger sequencing.

Engineering Homogeneous Photoactive Antibody Fragments 3.2.2 Cloning of Photocaged TyrosineSpecific Methanocaldococcus jannaschii Tyrosyl–tRNA Synthetase into pULTRA Suppressor Plasmid

29

1. Replace the MjCNFRS gene in the suppressor plasmid pULTRA-CNF with pcY-specific MjpcYRS. The mutant MjpcYRS has the following five mutations in comparison to the wild-type MjTyrRS: Y32G, L65G, F108E, D158S, and L162E [24]. Design a codon-optimized gene fragment for MjpcYRS, add the 25 base pair overhangs at the 50 - and 30 -ends of the designed gene fragment, and order this as a duplex DNA from a custom gene synthesis supplier (Fig. 1). We name this gene fragment as “MjpcYRS gene fragment.” 2. Remove the gene corresponding to MjCNFRS from pULTRACNF plasmid. In a 30 μL restriction digest reaction, incubate 1000 ng of pULTRA-CNF plasmid with 1 μL of 20,000 units/ mL NotI at 37  C for 1 h. Run the reaction mixture on 1% agarose gel and cut the band corresponding to the pULTRA_MjtRNACUA backbone (4 kb). Extract the pULTRA_MjtRNACUA vector backbone using a gel extraction kit. 3. Follow steps 4 to 10 in Subheading 3.2.1 for the subsequent operations.

3.2.3 Test Expression of Photocaged Tyrosine Containing Antibody Fragments

1. Transform electrocompetent BL21(DE3)pLysS cells with pSANG10 expression plasmid (containing gene for expression of amber mutant of antibody fragment), named pSANG10– antibody fragment plasmid, and pULTRA suppressor plasmid (containing genes for expression of Mj(pcY)RS and MjtRNACUA), named pULTRA-pcY plasmid. For this, add 1 μL each of pSANG10-antibody fragment plasmid (100 ng/μL) and pULTRA-pcY plasmid (100 ng/μL) to 50 μL of electrocompetent BL21(DE3)pLysS cells. Incubate on ice for 5 min and perform electroporation. 2. After electroporation, recover the cells by adding 500 μL of SOB media and incubating the cells at 37  C for 1 h, with shaking on a thermomixer at 900 rpm. 3. Plate 50 μL of recovered cells onto LB–agar plates supplemented with kanamycin 50 μg mL1 and spectinomycin 100 μg mL1. Incubate the plates overnight (37  C, 12–16 h). 4. Pick a single colony from this plate and inoculate 5 mL of 2xTY-GKS media. Incubate at 37  C and 220 rpm for 12–16 h. 5. The following day, subculture 30 mL 2xTY-GK1/2S1/2 media with the overnight culture such that OD600 ¼ 0.1. 6. Incubate at 37  C and 220 rpm for 2–3 h until OD600 ¼ 0.4 to 0.6, and then transfer 10 mL of this culture into two 50 mL falcon tubes (+ncAA and –ncAA). Add 10 μL of 1 M IPTG into both cultures for a final concentration of 1 mM. For the +ncAA culture, supplement the culture with 100 μL of 200 mM pcY stock solution for a final concentration of 2 mM pcY, and adjust the pH to 7 with 1 M HCl. Incubate these cultures at 30  C and 160 rpm for 12–16 h.

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7. The next day, record OD600 for both cultures and pellet the cells by centrifugation at 3200 g and 4  C for 10 min. Discard the supernatant and resuspend the pellet in 0.5 mL periplasmic extraction buffer 1. Incubate on ice for 30 min. 8. Centrifuge at 10,000 g and 4  C for 10 min. Transfer the supernatant to a fresh 1.5 mL tube and store at 4  C (periplasmic fraction 1). 9. Resuspend the pellet in 0.5 mL periplasmic extraction buffer 2 and incubate on ice for 20 min. 10. Centrifuge at 10,000 g and 4  C for 10 min. Transfer the supernatant to a fresh 1.5 mL tube and store at 4  C (periplasmic fraction 2). 11. Pool both periplasmic fractions together and centrifuge again at 10,000 g and 4  C for 10 min to remove any residual cell debris. Transfer the supernatant to a fresh 1.5 mL tube. 12. To purify antibody fragment using C-terminal His6 tag, add 50 μL of Ni-NTA resin to pooled periplasmic fractions. Mix gently on a rocker for 1 h at 4  C. 13. After incubation, pellet Ni-NTA resin by centrifugation at 300 g and 4  C for 1 min. Discard the supernatant (do not decant but remove the supernatant carefully without disturbing the resin bed and using a pipette). Wash resin with PBS by adding 1 mL of 1
PBS, mix by gently inverting the tube for a few times but do not vortex, centrifuge at 300 g and 4  C for 1 min, and discard the supernatant. Repeat PBS wash for another three times. 14. Next wash with Ni-NTA wash buffer. Follow the same procedure as above, this time with Ni-NTA wash buffer in place of 1
PBS. Repeat three times. 15. To elute the recombinant antibody fragment from the Ni-NTA resin, add 100 μL of SDS-PAGE sample buffer (1
Nu-PAGE LDS loading buffer containing 100 mM DTT), heat the sample to 95  C for 5 min, and centrifuge at 13,000 g and 4  C for 15 min. 16. Remove the supernatant and transfer it to a fresh tube. 17. Load 25 μL of supernatant containing the eluted antibody fragment on a 4–12% Bis–Tris gel, along with a protein standard ladder and run at 200 V for 40 min in MES SDS running buffer. 18. Stain the gel with Coomassie protein stain. Add 10 mL of the staining solution and leave the gel on a rocker for overnight. Destain the gel by washing with water. Image the gel using a gel imager. Figure 2 demonstrates site-specific incorporation of pcY at positions 32, 109, and 113 in an antibody fragment,

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Fig. 2 Expression of 7D12 mutants containing site-specifically incorporated pcY. wt7D12 and its three amber mutants, viz., 32TAG, 109TAG, and 113TAG, were expressed in the presence and absence of pcY. For amber mutants, a full-length protein is observed only in the presence of pcY, which is consistent with sitespecific incorporation of pcY in 7D12. (Reproduced from Ref. 8, which is an open-access article under the terms of the Creative Commons Attribution License)

7D12. For amber mutants, full length protein is observed for expression performed with pcY, demonstrating its site-specific incorporation in 7D12. Also, comparison of band intensities between wild-type 7D12 and its pcY-containing mutants demonstrates efficient incorporation of pcY (see Note 4). 3.2.4 Large Scale Expression of Photocaged Tyrosine Containing Antibody Fragments

1. Transform electrocompetent BL21(DE3)pLysS cells with pSANG10 expression plasmid (containing gene for expression of amber mutant of antibody fragment) and pULTRA suppressor plasmid (containing genes for expression of MjpcYRS and MjtRNACUA), as described in the previous section. After transformation, plate 50 μL of recovered cells onto LB–agar plates supplemented with 50 μg/mL kanamycin and 100 μg/mL spectinomycin. Incubate the plates overnight at 37  C for 16 h. 2. Pick a single colony from this plate and inoculate into 50 mL of 2xTY-GKS media. Incubate overnight at 37  C and 220 rpm for 12–16 h. 3. The following day, subculture 500 mL 2xTY-GK1/2S1/2 media with the overnight culture such that OD600 ¼ 0.1. 4. Incubate at 37  C and 220 rpm for 2–3 h until OD600 ¼ 0.4–0.6, then induce expression with 500 μL of 1 M IPTG (final concentration ¼ 1 mM). Supplement this culture with 5 mL of 200 mM pcY stock solution for a final concentration of 2 mM pcY, and adjust the pH to 7 by addition of 1 M HCl. Incubate this culture overnight at 30  C and 160 rpm for 12–16 h. 5. The next day, pellet the cells by centrifugation at 3200 g and 4  C for 10 min. Discard the supernatant and resuspend the cell pellet in 25 mL periplasmic extraction buffer 1 and incubate on ice for 30 min.

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6. Centrifuge at 10,000 g and 4  C for 10 min. Transfer the supernatant with a pipette to a sterile 50 mL tube and store at 4  C (periplasmic fraction 1). 7. Resuspend the cell pellet in 25 mL periplasmic extraction buffer 2 and incubated on ice for 20 min. 8. Centrifuge at 10,000 g and 4  C for 10 min. Transfer the supernatant with a pipette to a sterile 50 mL tube and store at 4  C (periplasmic fraction 2). 9. Pool both periplasmic fractions together and centrifuge again at 10,000 g and 4  C for 10 min to remove any residual cell debris. Transfer the supernatant to a fresh 50 mL tube. 10. To purify antibody fragment with Ni-NTA affinity chromatography, add 500 μL of Ni-NTA resin to the combined periplasmic extract and mixed gently on a rocker for 1 h at 4  C. 11. After incubation, transfer into a gravity-flow column (see Note 5) and wash three times with 1
PBS buffer, 10 mL each time, followed by three washes each with 10 mL of Ni-NTA wash buffer. 12. To elute the bound antibody fragment, add 500 μL Ni-NTA elution buffer and incubate for 15 min at room temperature, repeat elution step 8 times (see Note 6). Pool elution fractions together and dialyze overnight at 4  C against 1
PBS buffer. 13. Concentrate the dialyzed fractions using Vivaspin 500 columns with 3 kDa molecular weight cutoff. Determine the protein concentration using a colorimetric BCA protein assay. Dilute to the desired stock concentration and divide into small aliquots. Store aliquots at 20  C (see Note 7). 14. Analyze antibody fragment purity by resolving on SDS-PAGE. Prepare a 20 μL antibody fragment sample containing 0.5–3 μg purified protein, 1
Nu-PAGE LDS loading buffer and 1 mM DTT. Heat sample to 95  C for 5 min, followed by centrifugation at 13,000 g and 4  C for 15 min. 15. Load 20 μL samples on to a 4–12% Bis–Tris gel along with a protein ladder and run at 200 V for 40 min in MES SDS running buffer. 16. Stain the gel with Coomassie protein stain. Add 10 mL of the staining solution and leave the gel on a rocker for 8–14 h. Destain the gel by washing with water. Image the gel using a gel imager. 3.3 Light-Mediated Decaging of Photoactive Antibody Fragments

1. For decaging of the photocaged antibody fragments, place 10 μL of 50 μM caged antibody sample onto a 0.16 mm-thick glass coverslip (surface area of coverslip ¼ 2.54 cm2) or into a 96-well clear-bottom plate.

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Fig. 3 Mass spectrometry analysis of pcY-containing mutants of the antibody fragment, 7D12. The molecular weight of pcY-containing mutants of 7D12 decreases to that of wt7D12 upon irradiation with 365-nm light, demonstrating loss of o-nitrobenzyl group and complete decaging. (Reproduced from Ref. 8, which is an openaccess article under the terms of Creative Commons Attribution License)

2. Irradiate the sample with 365-nm light using a UV transilluminator for 2–10 min with a photon flux and the intensity of 33 mW/cm2 and 14 mW, respectively, at 365 nm (see Notes 8 and 9). 3. Analyze the decaged antibody samples using electrospray ionization mass spectrometry coupled with liquid chromatography (LC-ESI-MS). Figure 3 shows an example of LC-ESI-MS analysis of pcY-containing 7D12 mutants before and after irradiation with 365-nm light. 3.4 Measuring the Binding of (Photocaged) Antibody Fragments Against Antigens Expressed on the Surface of Live Cells

The next step involves assessing if the site-specifically incorporated photocaged tyrosine in the antibody fragment inhibits antibody– antigen interaction. Several methods are available to calculate antibody–antigen binding affinity, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), ELISA, flow cytometry, etc. [25–27]. The user can decide which method is most appropriate. Here, we describe an on-cell assay to measure the binding affinity of the antibody against an antigen expressed on the surface of cancer cells. This method (1) allows measurement of the binding affinity under biologically relevant conditions, i.e., in serum-containing media and towards antigen expressed on the cell surface, (2) does not require purified antigen, (3) does not require specialist equipment, and (4) is low cost and less resource intensive. This method has been validated to measure the binding affinity of 7D12 antibody fragment against its antigen, epidermal growth

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factor receptor (EGFR) expressed on human epidermoid carcinoma cell line A431. A similar experimental design can be applied to measure the binding affinity of other antibody fragments against target antigens expressed on the cell surface. Our on-cell assay starts with seeding antigen-expressing cells in a TC-treated 96-well plate. These cells are incubated with varying concentrations of the wild-type antibody and its photocaged mutants in DMEM media containing 10% serum in a 96-well plate. After incubation, unbound antibody is removed, cells are fixed to the surface, and antibody bound to the cell surface is estimated by detecting the hexa-histidine (His6) tag at the C-terminus of the antibody. 1. Grow A431 cells in a T-75 flask in complete medium (DMEM, 10% FBS, 1% PEN/STREP) using standard tissue culture procedures until 80–90% confluence. 2. Once the desired confluence has reached, remove and discard tissue culture medium from T-75 and gently wash cells with 10 mL of 1
DPBS. 3. Add 4 mL pre-warmed (37  C) trypsin to the flask to detach the cells from the surface. Gently rock the container to ensure complete coverage of the cell layer. 4. Incubate the T-75 flask in tissue culture incubator for 3 min at 37  C and 5% CO2. Note that the actual incubation time varies with the cell line used. After 3 min, tap vigorously at the bottom of T-75 to help detach cells. 5. Use a microscope to check for cell detachment. If less than 80–90% of cells are detached, increase incubation time by 30-s increments until cell detachment is complete. 6. Add 8 mL of pre-warmed (37  C) complete growth medium to trypsinized cells. For best recovery of cells, pipette growth medium over cell surface layer several times. 7. Transfer the cells to a 15 mL tube and centrifuge at 300 g for 5 min. 8. Gently remove and discard the supernatant leaving around 0.5 mL so as not to disturb the cell pellet. Subsequently add 10 mL of fresh pre-warmed complete medium and resuspend the cells. 9. Determine cell density and percent viability using a hemacytometer. Dilute the cell suspension to cell density of 200 cells/ μL in complete medium. Seed 200 μL of this cell suspension into each well of a 96-well white plate so that each well contains about 40,000 cells (see Note 10). Incubate this plate at 37  C and 5% CO2 for 12–16 h in a tissue culture incubator.

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10. The following day, prepare 100
stock solutions of purified antibody fragments in 1
PBS. For this, prepare a 96-well serial dilution clear plate. In each well, add 108.9 μL of complete medium and 1.1 μL of 100
stock antibody fragment (see Note 11). 11. Remove the 96-well white plate containing the cells after overnight incubation. Check under the microscope if the cells have attached to the surface of the wells. Remove the medium from wells using a multichannel pipette. Again, using a multichannel pipette, transfer 100 μL of pre-prepared medium supplemented with the antibody fragment from a serial dilution plate onto the cells. 12. Incubate this plate at 37  C and 5% CO2 for 5 min in a cell culture incubator. 13. If assessing the binding of photocaged antibody fragments after light-mediated activation, irradiate the plate with 365-nm light using the conditions described in Subheading 3.3. 14. After incubation (and irradiation if relevant), wash each well of the 96-well plate containing cells gently with 200 μL of fresh complete medium. 15. Remove the medium and fix cells using 150 μL of fixing solution. Incubate for 20 min at room temperature. 16. Remove fixing solution with a multichannel pipette discarding it in a suitable waste container. 17. Wash the wells three times with 150 μL assay wash buffer. For this incubate at room temperature with rocking for 5 min each time (see Note 12). 18. Add 100 μL of blocking solution to each well and incubate for 1 h at room temp with rocking. 19. Remove the blocking solution and wash once with 150 μL of wash buffer. 20. Add 50 μL of primary antibody solution to each well and incubate for 1 h at room temperature with rocking (see Note 13). 21. Wash the wells five times with 150 μL of wash buffer by incubating at room temperature with rocking for 5 min each time. 22. Add 50 μL of secondary antibody solution to each well and incubate for 1 h at room temperature with rocking (see Note 13). 23. Wash the wells ten times with 150 μL of assay wash buffer (room temperature with rocking for 5 min each time). Tap plate onto a clean paper towel before the first wash and after the last wash (see Note 14).

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24. Add 100 μL of chemiluminescent substrate to each well and incubate at room temperature for 10 min. 25. Image the plate using a gel imager with chemiluminescence measurement capability. Figure 4a shows an example of such an image for 7D12 mutants. For wt7D12, the chemiluminescent intensity increases with increase in concentration of 7D12 demonstrating binding of the wt7D12 antibody fragment to EGFR expressed on the surface of A431 cells. It also shows that site-specifically incorporated pcY at positions 32 and 113 in 7D12 antibody fragment inhibits its binding to EGFR, and the binding is restored upon irradiation with 365-nm light. 26. Next, quantify the chemiluminescence intensity from each well by measuring the chemiluminescence signal using a plate reader. To ensure reproducibility, on-cell experiments should be performed at least in triplicates. These experiments can be used to calculate the binding affinity values (KD) of the antibody fragment towards its corresponding antigen on the cell surface. To ensure that data between the plates could be compared, each plate should contain a standard, for example, the wild-type antibody fragment. Figure 4b shows a plot of chemiluminescence intensity with a concentration of wt7D12 and its mutants containing site-specifically incorporated pcY.

4

Notes 1. Note that photocaged lysine can also be genetically encoded into proteins expressed in E. coli [28]. Thus, in addition to the tyrosine residues, lysine residues in the antibody at the antibody–antigen interface may be targeted for mutagenesis to photocaged lysine. 2. Plasmids containing amber stop codon mutations can also be generated using site-directed mutagenesis from the plasmid containing the wild-type antibody fragment sequence. 3. The negative control should also be performed using the digested plasmid without the antibody gene fragment. If the number of colonies in the negative control plate is similar to that in the experiment plate with the digested plasmid and antibody gene fragment, it would suggest that (1) plasmid digestion was incomplete and should be repeated, (2) digestion buffers or times were not optimum, or (3) vector backbone and digested fragment were not fully separated on agarose gel before gel extraction. 4. The incorporation efficiency of pcY can vary with the site of incorporation. This could be due to the codon context of the amber stop codon. If the amount of pcY-containing antibody

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Fig. 4 The binding of pcY-containing mutants of 7D12 antibody fragment was measured towards EGFR on the surface of A431 cells. (a) For wt7D12 and 7D12-109pcY, chemiluminescent intensity increases with increase in concentration of 7D12, while for 7D12-32pcY and 7D12-109pcY near background chemiluminescence is observed before irradiation, demonstrating that pcY at positions 32 and 113 inhibits 7D12-EGFR binding. For all pcY-containing mutants, chemiluminescence intensity similar to that of wt7D12 is observed after irradiation with 365-nm light demonstrating restoration of antibody–antigen binding. (b) Chemiluminescence intensity was quantified using a plate reader and plotted against concentration of 7D12, where the X-axis is in log scale. The lines show the trace obtained after fitting the data to a sigmoidal nonlinear equation using GraphPad. Error bars represent the standard deviation from the mean chemiluminescence intensity calculated using three independent replicates. The binding affinity, KD, is the concentration of the 7D12 where chemiluminescence intensity is half of the maximum chemiluminescence intensity. KD values of wt7D12 and 7D12-109pcY, before irradiation, were estimated to be 23 (2.6) and 31 (1.5) nM, respectively. KD values of wt7D12, 7D12-32pcY, 7D12-109pcY, and 7D12-113pcY, after irradiation, were estimated to be 20 (1.8), 37 ( 2.6), 27 (1.6), and 38 (2.6) nM, respectively. (Reproduced from Ref. 8, which is an openaccess article under the terms of the Creative Commons Attribution License)

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fragment obtained is less than 10% of the wild-type antibody, the incorporation efficiency may be optimized by swapping two codons upstream and downstream of the amber stop codon with the corresponding synonymous codons. 5. Care should be taken when selecting the system used for purifying caged antibody fragments. Many automated protein purification instruments employ UV light for detecting the protein. Use of such light could decage the photocaged tyrosine in antibody fragments. We thus recommended using spin or gravity-flow columns for protein purification and detecting the protein using a small aliquot of the fractions collected. 6. Measure the OD280 of each eluted fraction and continue collecting samples until OD280 drops below 0.05. 7. Expression yields can vary between antibody fragments and depend on which tyrosine residue is mutated (i.e., codon context of the amber stop codon). For anti-EGFR antibody fragment, 7D12 (wild-type version), we reproducibly obtained ~10 mg of wt7D12 per liter of culture after purification. This yield dropped to 2–5 mg for pcY containing mutants of 7D12. 8. We use GelDocMega (BioSystematica) as the UV transilluminator. We measured the photon flux and the intensity of 365-nm light using a laser power meter (FieldMate, Coherent) at the surface of the transilluminator. The photon flux and the intensity of 365-nm light were found to be 33 mW/cm2 and 14 mW, respectively. 9. The time required for decaging the photocaged antibody fragment may vary with 365-nm radiation source and the composition of the buffer. We thus recommended optimizing the irradiation time by analyzing the antibody fragment (before and after irradiation) using mass spectrometry. 10. Due to the edge effect, it is recommended to leave outer wells blank. 11. For accurate transfer of 100 μL from the serial dilution plate, it is recommended to increase the volume to 110 μL to account for pipetting error. 12. Once the cells are fixed to the surface of the wells, wash buffer can be removed by inverting and tapping the plate. 13. This step can be done overnight at 4  C. 14. To ensure minimum background thorough washing should be performed after secondary antibody incubation. If background is too high when measuring plate signal, repeat washing using the wash buffer for another five times.

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Competing Interests AS and TB have filed a patent, application no: WO-2020193981A1, related to the research described in this book chapter. References 1. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, Wu HC (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27(1):1. https://doi.org/10. 1186/s12929-019-0592-z 2. Jovcevska I, Muyldermans S (2020) The therapeutic potential of nanobodies. BioDrugs 34(1):11–26. https://doi.org/10.1007/ s40259-019-00392-z 3. Ranallo S, Prevost-Tremblay C, Idili A, ValleeBelisle A, Ricci F (2017) Antibody-powered nucleic acid release using a DNA-based nanomachine. Nat Commun 8:15150. https://doi. org/10.1038/ncomms15150 4. Warram JM, de Boer E, Sorace AG, Chung TK, Kim H, Pleijhuis RG, van Dam GM, Rosenthal EL (2014) Antibody-based imaging strategies for cancer. Cancer Metastasis Rev 33(2–3): 809–822. https://doi.org/10.1007/s10555014-9505-5 5. Gunnoo SB, Finney HM, Baker TS, Lawson AD, Anthony DC, Davis BG (2014) Creation of a gated antibody as a conditionally functional synthetic protein. Nat Commun 5: 4 3 8 8 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / ncomms5388 6. Mahmood T, Yang PC (2012) Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4(9):429–434. https://doi.org/10. 4103/1947-2714.100998 7. Chen YS, Hong MY, Huang GS (2012) A protein transistor made of an antibody molecule and two gold nanoparticles. Nat Nanotechnol 7(3):197–203. https://doi.org/10. 1038/nnano.2012.7 8. Bridge T, Shaikh SA, Thomas P, Botta J, McCormick PJ, Sachdeva A (2019) Sitespecific encoding of photoactivity in antibodies enables light-mediated antibody-antigen binding on live cells. Angew Chem Int Ed Engl 58(50):17986–17993. https://doi.org/10. 1002/anie.201908655 9. Jedlitzke B, Yilmaz Z, Dorner W, Mootz HD (2020) Photobodies: light-activatable singledomain antibody fragments. Angew Chem Int Ed Engl 59(4):1506–1510. https://doi.org/ 10.1002/anie.201912286 10. Yu D, Lee H, Hong J, Jung H, Jo Y, Oh BH, Park BO, Heo WD (2019) Optogenetic

activation of intracellular antibodies for direct modulation of endogenous proteins. Nat Methods 16(11):1095–1100. https://doi. org/10.1038/s41592-019-0592-7 11. Self CH, Thompson S (1996) Light activatable antibodies: models for remotely activatable proteins. Nat Med 2(7):817–820 12. Dumas A, Lercher L, Spicer CD, Davis BG (2015) Designing logical codon reassignment – expanding the chemistry in biology. Chem Sci 6(1):50–69. https://doi.org/10.1039/ c4sc01534g 13. Young DD, Schultz PG (2018) Playing with the molecules of life. ACS Chem Biol 13(4): 8 5 4 – 8 7 0 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 1 / acschembio.7b00974 14. Chin JW (2014) Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83:379–408. https://doi. org/10.1146/annurev-biochem060713-035737 15. de la Torre D, Chin JW (2021) Reprogramming the genetic code. Nat Rev Genet 22(3): 169–184. https://doi.org/10.1038/s41576020-00307-7 16. Bannas P, Hambach J, Koch-Nolte F (2017) Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol 8:1603. https://doi.org/ 10.3389/fimmu.2017.01603 17. Bates A, Power CA (2019) David vs. Goliath: the structure, function, and clinical prospects of antibody fragments. Antibodies (Basel) 8(2). https://doi.org/10.3390/antib8020028 18. Asaadi Y, Jouneghani FF, Janani S, Rahbarizadeh F (2021) A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomark Res 9(1):87. https://doi.org/10.1186/s40364-02100332-6 19. Yang EY, Shah K (2020) Nanobodies: next generation of cancer diagnostics and therapeutics. Front Oncol 10:1182. https://doi.org/ 10.3389/fonc.2020.01182 20. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A, Bridgland A, Meyer C, Kohl

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SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596(7873):583–589. https://doi.org/10.1038/s41586-02103819-2 21. Martin CD, Rojas G, Mitchell JN, Vincent KJ, Wu J, McCafferty J, Schofield DJ (2006) A simple vector system to improve performance and utilisation of recombinant antibodies. BMC Biotechnol 6:46. https://doi.org/10. 1186/1472-6750-6-46 22. Biffi G, Tannahill D, McCafferty J, Balasubramanian S (2013) Quantitative visualization of DNA G-quadruplex structures in human cells. Nat Chem 5(3):182–186. https://doi.org/10. 1038/nchem.1548 23. Chatterjee A, Sun SB, Furman JL, Xiao H, Schultz PG (2013) A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry 52(10):1828–1837. https://doi.org/10. 1021/bi4000244 24. Deiters A, Groff D, Ryu Y, Xie J, Schultz PG (2006) A genetically encoded photocaged

tyrosine. Angew Chem Int Ed Engl 45(17): 2728–2731. https://doi.org/10.1002/anie. 200600264 25. Bocker JK, Dorner W, Mootz HD (2019) Light-control of the ultra-fast Gp41-1 split intein with preserved stability of a genetically encoded photo-caged amino acid in bacterial cells. Chem Commun (Camb) 55(9): 1287–1290. https://doi.org/10.1039/ c8cc09204d 26. Jiang W, Cossey S, Rosenberg JN, Oyler GA, Olson BJ, Weeks DP (2014) A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components. BMC Plant Biol 14:244. https://doi.org/10. 1186/s12870-014-0244-0 27. Freyer MW, Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 84:79–113. https://doi.org/10. 1016/S0091-679X(07)84004-0 28. Gautier A, Deiters A, Chin JW (2011) Lightactivated kinases enable temporal dissection of signaling networks in living cells. J Am Chem Soc 133(7):2124–2127. https://doi.org/10. 1021/ja1109979

Chapter 3 Repurposing Photosensitizer Proteins Through Genetic Code Expansion to Facilitate Photo-Biocatalysis Jiangyun Wang, Yan Xia, and Xuzhen Guo Abstract Artificial photoenzymes with noncanonical photo-redox cofactors have paved the way for enzyme rational design and the creation of new-to-nature biocatalysts. Genetically encoded photo-redox cofactors endow photoenzymes with enhanced or novel activities that catalyze numerous transformations with high efficiency. Herein, we describe a protocol of repurposing photosensitizer proteins (PSP) through genetic code expansion to facilitate multiple photocatalytic conversions including photo-activated dehalogenation of aryl halides, CO2 to CO and CO2 to formic acid reduction. The methods for expression, purification, and characterization of the PSP are detailed. The installation of the catalytic modules and the utilization of PSP-based artificial photoenzymes for photoenzymatic CO2 reduction and dehalogenation are also described. Key words Artificial photoenzyme, Photosensitizer proteins, Genetic codon expansion, Photobiocatalysis

1

Introduction Nature has evolved tremendous protein enzymes catalyzing a remarkable range of chemical reactions in a mild, spatiotemporally controllable condition with only 20 natural amino acid building blocks and natural cofactors [1, 2]. Although there are many proteins that can accept photons from sunlight, surprisingly rare natural photoenzymes have been found. Natural photoenzymes mainly use chlorophyll-like tetrapyrrole or flavins as photo-redox cofactors for photo-biocatalysis [3]. Such examples include chlorophyllbased photoenzymes in the photo-driven biosynthesis of chlorophyll [4], flavin-based DNA photolyases that repair UV-induced DNA photolesions [5], and recently discovered flavin-based fatty acid photo-decarboxylases in microalga that metabolize fatty acids

Yan Xia and Xuzhen Guo contributed equally to this work Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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to the corresponding alkanes or alkenes [6, 7]. Due to the limited availability of natural photo-redox cofactors or photoenzymes, engineered and artificial photoenzymes have been created for new-to-nature photo-biotransformation, which undoubtedly fills the gaps and greatly expands the applications that surpass their natural counterparts [8, 9]. The photosensitizer is an important building block in photobiocatalysis as it initiates photo-induced charge separation [10]. Nowadays, many different types of photosensitizers have been developed to construct artificial photoenzymes. The inorganic nanomaterials (e.g., carbon dots, CdS nanoparticles, etc.) either contain heavy metals or have poor biocompatibility [11– 15]. On the other hand, organometallics (e.g., Ru(bpy)3, Ir (bpy)3, etc.) or organic photosensitizers (e.g., Eosin Y, Rose Bengal, etc.) need complicated conjugation with the protein scaffolds [16–18]. Besides that, most natural photosensitizer cofactors are small molecules that have proved difficult to enhance or expand their functions through genetic engineering. Genetic incorporation of noncanonical amino acids (ncAAs) provides a powerful tool for exploring protein structure and function, as well as gives proteins with enhanced or new properties [19– 21]. These ncAAs, containing metal-chelating, redox-active, and unique chemical or photochemical property, open the door for artificial enzyme design and engineering [22–24]. Benzophenone is a commonly used organic photo-redox catalyst that undergoes rapid intersystem crossing with nearly 100% efficiency from the singlet excited state to the triplet state after photo-activation [25]. The photo-redox noncanonical amino acid, benzophenonealanine (BpA), has been successfully incorporated into Escherichia coli [26], Saccharomyces cerevisiae [27], and mammalian cells [28] with specificity and fidelity through genetic codon expansion. The genetically encoded BpA has been utilized as a privileged photocrosslinker to map ligand-protein binding sites [29], to assist epitope-directed antibody selection [30], and to capture proteinprotein interactions [31, 32]. Artificial photoenzymes incorporated with BpA as the photo-redox cofactor have been repurposed to efficiently catalyze many photoenzymatic transformations, including enantioselective [2 + 2]-cycloaddition [33, 34], dehalogenation and C–N bond formation [35], and CO2 reduction to CO or formic acid [36, 37]. The green fluorescent protein (GFP) was first isolated from the jellyfish Aequorea victoria and now becomes a widely used fluorescent reporter in bioimaging [38]. The fluorescent protein features a barrel formed with 11 β-sheets and accommodates an internal distorted helix inside it [39]. The fluorophore in superfolder yellow fluorescent protein (sfYFP) [40] is endowed with autocatalytic maturation within the tripeptide (Gly65-Tyr66-Gly67) to form a highly fluorescent p-hydroxybenzylidene-5-imidizolinone species

Repurposing Photosensitizer Proteins Through Genetic Code Expansion. . .

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Fig. 1 Chemical structures of the photosensitizer noncanonical amino acid BpA (left) and the autocatalytically matured chromophore BpC (right) in PSP2 protein

[41]. We envisioned that, when the photo-redox BpA is genetically incorporated into sfYFP and replacing its Tyr66 residue, the tripeptide Gly65-BpA66-Gly67 can also be auto-maturated into the BpC chromophore that shares common features except for the benzophenone photosensitizer group (Fig. 1). After rational design and rounds of optimization, we have successfully obtained the photosensitizer protein named PSP2 [36], whose structure, photophysical, and photochemical properties have been further confirmed by mass spectrometry, X-ray crystallography, and transient absorption spectroscopy. This artificial protein PSP2 inherits the photochemical properties of benzophenone and has a long-lived triplet state that can generate a PSP2 radical after photoexcitation in the presence of a sacrificial reductant. The reduction potential of a PSP2 radical is near -1.5 V, which is lower than the known natural biological reductant to our knowledge. Due to the BpC chromophore being protected by the hydrophobic barrel of the PSP2 protein, the generated PSP2 radical is stable for more than 10 min in the absence of oxygen. Furthermore, a molecular catalyst nickelterpyridine complex [42] can be covalently tagged to the specific cysteine residue on the surface of the PSP2 protein, and the catalyst can accept the high-energy electron transferred from the BpC chromophore under photoirradiation, driving the reduction of CO2 to CO with a photo quantum yield at 2.6%, which is higher than most reported photo-driven CO2 reduction catalysts. Followed by the same strategy, a nickel-bipyridine complex [43] can be conjugated to the PSP2 protein at Cys95 residue to generate an artificial photo-dehalogenase [35], which can efficiently catalyze the dehalogenation of diverse aryl halides to phenols, or propel valuable C–N bond formation under photoexcitation. In addition, the efficiency can be significantly improved with precise control of the spatial distance between the BpC chromophore and the catalytic module. Besides organometallic catalysts, the PSP2 protein can also accommodate metalloprotein to reprogram CO2 reductase. Ferredoxin from Clostridium acidurici (CaFD) [44] which contains two [4Fe-4S] clusters can be genetically inserted into the PSP2 protein as the catalytic module. After fine-tuning the reduction potential of CaFD and optimizing the distance, multistep

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Fig. 2 Repurposing photosensitizer proteins through genetic code expansion to facilitate photo-biocatalysis

electron transfer process within the BpC chromophore and [4Fe-4S] clusters, the artificial photo-driven CO2 reductase mPCE achieved CO2 reduction to formic acid with a quantum efficiency of 1.43% [37]. Photosensitizer proteins stand out among many types of artificial photoenzymes because they are easier to construct and suitable for many photo-biotransformations with the simple replacement of the catalytic modules. They are more environment-friendly due to the lack of noble or heavy metals in comparison to inorganic-hybrid artificial photoenzyme systems. The PSP2 protein and the corresponding photoenzymes are genetically encoded and have great potential to achieve whole-cell photo-biocatalysis in living cells (Fig. 2). Herein, we detail our methods to generate a PSP2 protein from E. coil cells through genetic code expansion, and we highlight the techniques for protein purification and catalytic module installation within the PSP2 protein. A brief characterization of the properties and the utilization of the PSP2 protein for photodriven CO2 reduction and dehalogenation is also included.

2

Materials

2.1 Reagents, Cell Strains, and Plasmids

1. Ferrous ammonium sulfate FeSO4·(NH4)2SO4·6H2O, CAS: 7783-85-9.

hexahydrate,

2. Hydrochloric acid, HCl, CAS: 7647-01-0. 3. Nickel perchlorate hexahydrate, Ni(ClO4)2·6H2O, CAS: 13520-61-1. 4. Nickel sulfate hexahydrate, NiSO4·6H2O, CAS: 10101-97-0. 5. Sodium bicarbonate, NaHCO3, CAS: 144-55-8. 6. Sodium chloride, NaCl, CAS: 7647-14-5.

Repurposing Photosensitizer Proteins Through Genetic Code Expansion. . .

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7. Sodium dithionite, Na2S2O4, CAS: 7775-14-6. 8. Sodium hydroxide, NaOH, CAS: 1310-73-2. 9. Sodium sulfide, Na2S, CAS: 1313-82-2. 10. 2,3-Dihydro-1,3-dimethyl-2-phenyl-1H-benzimidazole (BIH), CAS: 3652-92-4. 11. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic (HEPES), CAS: 7365-45-9.

acid

12. Acetic acid, CAS: 64-19-7. 13. Benzophenone-alanine (BpA), CAS: 104504-45-2. 14. Carbenicillin disodium, CAS: 4800-94-6. 15. Chloramphenicol, CAS: 56-75-7. 16. Diisopropylethylamine (DIPEA), CAS: 7087-68-5. 17. Dimethylformamide (DMF), CAS: 68-12-2. 18. Dithiothreitol (DTT), CAS: 7634-42-6. 19. Ethanol, CAS: 64-17-5. 20. Ethyl acetate, CAS: 141-78-6. 21. Ethylenediaminetetraacetic acid (EDTA), CAS: 60-00-4. 22. Imidazole, CAS: 288-32-4. 23. Isopropyl β-D-thiogalactopyranoside (IPTG), CAS: 367-93-1. 24. L-Arabinose, CAS: 5328-37-0. 25. L-Ascorbic acid, CAS: 50-81-7. 26. Tris(hydroxymethyl)aminomethane (Tris), CAS: 77-86-1. 27. Tris(2-carboxyethyl)phosphine (TCEP), CAS: 5961-85-3. 28. N-(2,6,2-terpyridine-4-yl)-iodoacetamide. 29. 3-kDa and 10-kDa ultrafiltration tubes for concentrating protein samples. 30. Argon gas cylinder. 31. E. coli Top10 and BL21(DE3) cell strains for plasmid cloning and protein expression. 32. Ni-NTA agarose resin for His-tagged protein purification. 33. Plasmid pEVOL-BpARS, available through Addgene (Plasmid #31190). 34. Chromatography columns: Superdex 75 10/300 GL, Sephadex G25. 35. Kit for Bradford assay for quantifying protein concentration. 36. Quartz cuvette for UV-visible spectrophotometer. 37. Ultrapure water system.

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Instruments

1. Fast protein liquid chromatography (FPLC) for protein purification. 2. UV-visible spectrophotometer. 3. Ultrasonic crusher. 4. High-performance liquid chromatography-mass spectrometry (HPLC-MS). 5. Gas chromatography (GC). 6. Xe lamp and UVCUT400 filter. 7. 380-nm LED. 8. Nuclear magnetic resonance (NMR) spectrometer.

2.3 Culture Medium, Stock Solution, and Buffers

1. LB broth: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. Sterilization by autoclaving at 15 psi for 20 min. 2. Carbenicillin stock solution: 100 mg/mL ddH2O. Sterilization through a 0.22 μm syringe filter.

in

3. Chloramphenicol stock solution: 50 mg/mL in ethanol. 4. BpA solution: 100 mM in 200 mM NaOH solution. 5. L-Arabinose stock solution: 20% (w/v) in ddH2O. Sterilization through a 0.22 μm syringe filter. 6. IPTG stock solution: 1 M in ddH2O. Sterilization through a 0.22 μm syringe filter. 7. Lysis buffer: 50 mM Tris–HCl at pH 7.5, 150 mM NaCl, and 10 mM imidazole. 8. Wash buffer: 50 mM Tris–HCl at pH 7.5, 150 mM NaCl, and 50 mM imidazole. 9. Elution buffer: 50 mM Tris–HCl at pH 7.5, 150 mM NaCl, and 500 mM imidazole. 10. HEPES buffer: 20 mM HEPES at pH 7.5 and 200 mM NaCl. Sterilization through a 0.22 μm syringe filter and sonication for 10 min to remove air bubbles. 11. Protein storage buffer: 50 mM Tris–HCl, pH 7.5, and 150 mM NaCl. 12. Non-salt buffer: 10 mM Tris–HCl at pH 8. 13. Labeling buffer: 150 mM Tris–HCl at pH 8.8 in 30% (v/v) DMF to increase the solubility of the organometallic catalysts. 14. Reaction buffer A: 100 mM Tris–HCl at pH 8.0 in 50% (v/v) DMF to increase the solubility of the sacrificial reductant. 15. Reaction buffer B: 75 mM Tris–HCl at pH 8.8 in 5% or 25% (v/v) DMF depending on specific substrate.

Repurposing Photosensitizer Proteins Through Genetic Code Expansion. . .

3

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Methods

3.1 Protein Expression Through Genetic Code Expansion 3.1.1 Plasmid Construction

3.1.2 Cell Culture and Protein Expression

Clone the gene encoding the PSP2 protein into the pET22b vector (see Note 1). Use pET22b-PSP2 as the template for generating different PSP2 mutants by site-specific mutagenesis. Confirm all the mutants by Sanger sequencing. Insert ferredoxin CaFD (see Note 2) into the loop (position 37–40) of the PSP2 protein. Remove Thr38 and Ile39 to accommodate the insertion of CaFD. Insert the gene encoding CaFD into the PSP2 gene through overlap PCR and confirm the identity of plasmid pET22b-mPCE by Sanger sequencing. 1. Co-transform plasmids pET22b-PSP2 (or pET22b-mPCE) and pEVOL-BpARS into E. coli BL21(DE3) cells (see Note 3). 2. Culture the cells overnight at 37 °C and 220 rpm in 4 mL of LB medium supplemented with 100 μg/mL carbenicillin and 25 μg/mL chloramphenicol. 3. Use the overnight culture to inoculate 400 mL of LB medium. Keep this culture at 37 °C and 220 rpm until OD600 = 0.8–1. Add reagents to the final concentration of 0.02% (w/v) arabinose, 1 mM IPTG, and 1 mM BpA (see Note 4) to induce protein expression. 4. Culture the cells for another 6–8 h at 37 °C and 220 rpm. Harvest the cells by centrifugation and store the pellets at 80 °C (see Note 5).

3.2 Protein Purification

The consumption of the buffers is based on cells harvested from 400 mL culture medium. A cool environment should be ensured to keep the protein in its optimal state throughout the purification process (see Note 6). 1. Resuspend the cells in 8 mL lysis buffer. Disrupt the cells by sonication (200 W, 5-s on and 5-s off for a total of 30 min; see Note 7). 2. Separate the protein from the cell debris by centrifugation at 13,000 g and 4 °C for 30 min. Load the supernatant onto a Ni-NTA agarose resin and keep for 1 h to ensuring binding of the target protein. 3. Wash the Ni-NTA resin with 25 mL washing buffer twice and then elute the protein with 5 mL elution buffer (see Note 8). 4. Concentrate the protein using a 10-kDa ultrafiltration tube to a final volume of approximately 500 μL (see Note 9). 5. Further purify the protein by size-exclusive chromatography using a FPLC at 4 °C. 6. Quantify the protein concentration using the Bradford assay. The yield of the desired protein should be about 20 mg/L.

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3.3 Characterization of the Proteins 3.3.1

UV–Vis Spectra

The PSP and the PSP radical both have specific absorption peaks in the visible light region. To verify the generation of a PSP radical, irradiate 50 μM purified PSP2 with a 405-nm laser in the presence of 10 mM dithionite or 100 mM ascorbate. Record the UV–vis spectra using a quartz cuvette (100 μL, 1 cm path) at room temperature with UV–visible spectrophotometer (see Note 10).

3.3.2 Mass Spectrometry Analysis

Mass spectrometry analysis can be used to determine the molecular weight of the purified proteins, to verify the successful insertion of BpA, and to identify the modification of different catalyst conjugates. In our case, dilute the protein samples to 50 μg/mL in ddH2O before analysis by HPLC-MS.

3.4 Artificial Photoenzyme for CO2/ CO Conversion

As for photo-activated CO2 reduction, coupling a small-molecule catalyst, Ni(terpyridine), to the PSP2 protein affords a miniature photocatalytic CO2-reducing enzyme that has a CO2/CO conversion quantum efficiency of 2.6% with the following experimental protocol.

3.4.1 Terpyridine Modification of SingleCysteine PSP2 Mutant

1. Dissolve the single-cysteine PSP2 mutant (see Note 11) in the labeling buffer to the final concentration of 50 μM. React with 100 μM TCEP for 5 min at room temperature to release the thiol group of cysteine. 2. Add 250 μM N-(2,6,2-terpyridine-4-yl)-iodoacetamide into the labeling mixture. Incubate the reaction at room temperature for another 12 h. 3. Remove the excess terpyridine using a 0.5 mL 3-kDa ultrafiltration tube and then concentrate the protein into the non-salt buffer. 4. Analyze the modified product by HPLC-MS.

3.4.2 Photo-Activated CO2/CO Conversion with PSP2-Ni(Terpyridine)

Perform the photocatalytic CO2/CO conversion with artificial PSP2-Ni(terpyridine) in a 10 mL glass bottle sealed with a crimp cap (see Note 12). 1. Prepare the reaction with the total volume of 200 μL containing 80 μM Ni(ClO4)2, 100 mM NaHCO3, and 100 mM BIH in reaction buffer A and 40 μM PSP2-(terpyridine) (see Note 13). 2. Remove the oxygen by bubbling with argon for 10 min and then seal the reaction vessel with a crimp cap. 3. Irradiate the reaction mixture using a 300 W Xe lamp with an AM 1.5 filter to mimic the solar spectrum. Use a cutoff filter (UVCUT400) to achieve visible-light (λ > 400 nm) irradiation (see Note 14). 4. Analyze the generated gas and its evolution rate by gas chromatography.

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3.5 Artificial Photoenzyme for CO2/ HCOOH Conversion

The miniature photocatalytic CO2-reducing enzyme (mPCE) is designed by fusing ferredoxin CaFD with the PSP2 protein [37]. Through fine-tuning the reduction potential of CaFD, we optimize the multiple-step electron hopping from the BpC chromophore to FeA/FeB clusters and achieve a CO2/HCOOH conversion quantum efficiency of 1.43% with the following experimental protocol.

3.5.1 Reconstitution of the [4Fe-4S] Clusters in mPCE

1. Perform all reconstitution steps under an argon atmosphere in a glove box. First, exchange the buffer of mPCE protein to the non-salt buffer before reconstitution. Add DTT to a final concentration of 5 mM. Incubate the mixture at room temperature for 5 min. 2. Add ammonium iron (II) sulfate and sodium sulfide into the mixture each to a final concentration of 1 mM. Incubate the reaction mixture at room temperature for another 4–6 h. A dark brown color should gradually appear. 3. After the incubation, add 2 mM EDTA into the reaction mixture to extract free iron ions. 4. Remove precipitates and Fe-S particulates in the reaction mixture by centrifugation. Remove excess reagents through sizeexclusive chromatography using a Sephadex G25 column and the storage buffer.

3.5.2 Photo-Activated CO2/HCOOH Conversion with mPCE

Conduct the photoenzymatic CO2 reduction with mPCE in a 10 mL glass bottle sealed with a crimp cap. 1. Prepare a reaction of about 500 μL containing 100 mM NaHCO3 and 100 mM sacrificial reductant BIH in reaction buffer A, followed by addition of 40 μM mPCE protein. 2. Remove the oxygen by bubbling with argon for 10 min and then seal the reaction vessel with a crimp cap. Irradiate the reaction mixture using a 300 W Xe lamp with an AM 1.5 filter to mimic the solar spectrum. Use a cutoff filter (UVCUT400) to achieve visible-light (λ > 400 nm) irradiation. 3. Add D2O to the reaction mixture to a final concentration of 10% (v/v). Analyze the product by 1H and 13C NMR with 5 mM acetic acid as the standard.

3.6 Photo-Activated Dehalogenation with PSP2-Ni(Bipyridine)

Taking advantage of the strong reducing properties of the PSP radical, PSP2-Ni(bipyridine) can catalyze dehalogenation of diverse aryl halides to phenols with the following experimental protocol. Follow Subheading 3.4.1 for preparation of PSP2-Ni (bipyridine). Perform the photocatalytic dehalogenation of aryl halides with artificial PSP2-Ni(terpyridine) in a 10 mL glass bottle sealed with a crimp cap.

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1. Prepare the reaction with the total volume of 2 mL containing 0.1 μM NiSO4, 10 μM aryl halides (pre-dissolved in DMF), and 1.5 eq. sacrificial reductant DIPEA (2.5 μL) in reaction buffer B, followed by the addition of the bipyridine-modified PSP2 protein to the final concentration of 44 nM in a glove box and argon atmosphere. 2. Irradiate the reaction mixture with a 380-nm LED light source for 12 h. 3. Extract the product with ethyl acetate (7 × 3 mL). Concentrate the organic layers in vacuum to give the crude product, followed by analysis with 1H NMR (see Note 15).

4

Notes 1. The amino acid sequence of PSP2 is shown below with * indicating the site of BpA: MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLG *G LQCFAR YPDHMKQHDFFKSAMPEGYVQERTISFKDDGKYK TRAVVKFEGDTLVNRIELKGTDFKEDGNILGHK LEYNFNSENVYITADKQKNGIKANFTVRHN VEDGSVQLADHYQQNTPIGDGPVLLPDN HYLSDQTVLSKDPNEKRDHMVLLEFVTAAGITLGM DELYK. 2. The amino acid sequence of CaFD is shown below: AYVINEACISCGACEPECPVNAISSGDDRYVIDADTCIDCGACAGVCPVDAPVQA. 3. Plasmid pEVOL-BpARS harbors orthogonal M. jannaschii tyrosyl-tRNA synthetase and M. jannaschii tyrosyl amber suppressor tRNA (MjtRNATyrCUA) pair to allow for the sitespecific incorporation of BpA into the target protein. 4. As BpA is prepared in NaOH solution, it is important to shake the medium immediately to avoid affection of the cell vitality. If a large amount of NaOH solution is added, it should be neutralized with an equal molar amount of hydrochloric acid. 5. If the protein will be purified immediately after harvesting, there is no need to store the cells in -80 °C. 6. Even though the PSP is stable, it is better to keep the purification process at low temperate such as 4 °C. 7. To ensure complete disruption of the cells, it is not advisable to use large beakers. The ultrasonic disrupter should be in the abdominal position of the cell-resuspended mixture. High-

Repurposing Photosensitizer Proteins Through Genetic Code Expansion. . .

pressure crushing purification.

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recommended

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protein

8. For better elution of the target protein, it is important to ensure suitable binding time of the protein to the resin. Avoid violent shaking to decrease protein denaturation. 9. Buffer exchanging is necessary to remove excess imidazole in the protein solution. Protein leakage can be observed from the color of the protein in the tube bottom. The interval time between each buffer exchanging process should be as short as possible. Too long interval time may lead to protein precipitation due to very high concentration of the protein. 10. Because molar extinction coefficient differs among proteins, it is better to ensure the absorbance value is in the range of 0.1–1. 11. The amino acid sequence of single-cysteine PSP2 mutant is shown below with * indicating the site of BpA: MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLG *G LQCFAR YPDHMKQHDFFKSAMPEGYVQCRTISFKDDGKYK TRAVVKFEGDTLVNRIELKGTDFKEDGNILGHK LEYNFNSENVYITADKQKNGIKANFTVRHN VEDGSVQLADHYQQNTPIGDGPVLLPDN HYLSDQTVLSKDPNEKRDHMVLLEFVTAAGITLGM DELYK. 12. The radical reactions must be conducted in an anaerobic environment as oxygen can quench free radicals. 13. Add water and buffer first, followed by small molecules and finally the protein. Mix the reaction system after each addition. 14. The light intensity varies greatly with distance and position. The light intensity should be determined before each photoirradiation experiment for subsequent quantum yield calculation. 15. The yield of dehalogenation products is generally calculated based on the integration of the starting material and product in 1 H NMR.

Acknowledgments The authors are grateful to the National Key R&D Program of China (2021YFA0910802, 2019YFA0904002, 2019YFA0904103, 2020YFA0908503, 2020YFA0907701) and Sanming Project of Medicine in Shenzhen (no. SZSM201811092).

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Chapter 4 Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities Manjia Li and Tao Peng Abstract The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Fo¨rster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for sitespecific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying the activities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases. Key words Noncanonical amino acid, Fluorescent amino acid, Genetic code expansion, Deubiquitinase, FRET pair

1

Introduction The genetic code expansion technology [1, 2] has provided a unique and powerful strategy for fluorescent labeling of proteins with site specificity and atomic resolution [3, 4]. This is normally achieved through a two-step process. Bioorthogonal noncanonical amino acids (ncAAs) are first incorporated into the proteins of interest, followed by bioorthogonal conjugations to site-specifically attach small-molecule fluorophores [5–10]. Nevertheless, a series of fluorescent ncAAs [11–19] have been developed and sitespecifically incorporated into proteins at defined positions [4, 20], providing a straightforward solution for direct, one-step fluorescent labeling. Owing to the small size, genetic encodability, and internal labeling site, fluorescent ncAAs are particularly useful in constructing two-fluorophore cassette probes that are capable of Fo¨rster resonance energy transfer (FRET) [21], in which a donor fluorophore transfers energy to an acceptor fluorophore. Indeed,

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Genetic encoding of an aminocoumarin-derived fluorescent noncanonical amino acid for the analysis of deubiquitinase activities. (a) Structure of the aminocoumarin-derived fluorescent amino acid, AFCouK. (b) Schematic of the AFCouK-based fluorescent probe, UbAFCouK-sfGFP, for detecting deubiquitinase activities. AFCouK at the internal site of ubiquitin forms a FRET pair with the C-terminal sfGFP, whereas deubiquitinases cleave ubiquitin and disrupt the FRET, leading to increases of AFCouK fluorescence (λem = 440 nm) and decreases of sfGFP fluorescence (λem = 510 nm). (Reproduced from Ref. [19] with permission from the Royal Society of Chemistry)

many fluorescent ncAA-based FRET probes have been reported for studying protein structural changes [15, 22–24], interactions [25– 27], and stability [28]. Deubiquitinases are a large group of ubiquitin-specific proteases that cleave ubiquitin from substrate proteins [29]. Deubiquitinases have been implicated in many diseases including cancer and neurodegeneration and therefore represent potential drug targets [30]. Fluorescent probes, such as coumarin- or rhodamineconjugated ubiquitin [31, 32] and FRET-based diubiquitin [33], have been developed for profiling deubiquitinase activities [34]. However, these probes require chemical labeling and/or chemical synthesis of the ubiquitin moieties. Recently, we reported the genetic encoding of an aminocoumarin-derived fluorescent ncAA, N6-((2-(7-amino-6-fluoro-2-oxo-2H-chromen-4-yl)ethoxy)carbonyl)-L-lysine (i.e., AFCouK, Fig. 1a), and its application as the FRET donor to construct a fully genetically encoded fluorescent probe, ubiquitin-Y59AFCouK-sfGFP (UbAFCouK-sfGFP), for deubiquitinases [19]. Specifically, AFCouK incorporated at the internal site of ubiquitin forms an efficient FRET pair with the C-terminal superfolder green fluorescent protein (sfGFP), whereas deubiquitinases cleave ubiquitin and disrupt the FRET, leading to increases of AFCouK fluorescence (λem = 440 nm) and decreases of sfGFP fluorescence (λem = 510 nm) (Fig. 1b). In this protocol, we describe the experimental steps for the sitespecific incorporation of AFCouK into proteins in E. coli using an engineered Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS), i.e., ACouKRS, that can specifically aminoacylate its cognate pyrrolysyl-tRNA (tRNAPyl) with AFCouK. We also detail the preparation of the UbAFCouK-sfGFP probe and an in vitro UbAFCouK-sfGFP-based fluorescence assay to examine the activities of deubiquitinases and screen small-molecule inhibitors against deubiquitinases.

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2.1 ncAA and Plasmids

1. Dimethyl sulfoxide (DMSO). 2. AFCouK stock solution (500 mM): Dissolve 0.246 g of AFCouK in 1 mL of DMSO; store at 4 °C (see Note 1). 3. pACouKRS plasmid (see Note 2). 4. pEGFP-Y39TAG plasmid (see Note 3). 5. pUb-Y59TAG-sfGFP plasmid (see Note 4).

2.2 Site-Specific Incorporation of AFCouK into Proteins in E. coli

1. E. coli strain BL21(DE3) chemically component cells. 2. Kanamycin stock solution (1000×): Dissolve 40 mg of kanamycin sulfate in 1 mL of ultrapure water and sterilize with a 0.2 μm syringe filter. 3. Chloramphenicol stock solution (1000×): Dissolve 34 mg of chloramphenicol in 1 mL of absolute ethanol and sterilize with a 0.2 μm syringe filter. 4. LB medium: Dissolve 8 g of tryptone, 8 g of NaCl, and 4 g of yeast extract in 800 mL of ultrapure water and autoclave for 20 min at 120 °C; store at room temperature. 5. LB agar plates supplemented with kanamycin and chloramphenicol: Mix 8 g of tryptone, 8 g of NaCl, 4 g of yeast extract, and 12 g of agar in 800 mL of ultrapure water and autoclave for 20 min at 120 °C; add kanamycin and chloramphenicol stock solutions (each 0.8 mL) when cooled to ~50 °C and pour into Petri dishes. 6. 1 M IPTG stock solution: Dissolve 238 mg of isopropyl-β-Dthiogalactopyranoside in 1 mL of ultrapure water and sterilize with a 0.2 μm syringe filter; store at 4 °C. 7. SDS lysis buffer: 4% SDS, 150 mM NaCl, 50 mM triethanolamine, pH 7.4. 8. Precast polyacrylamide gels for SDS-PAGE. 9. SDS-PAGE running buffer: Dissolve 6.06 g of Tris-base, 10.46 g of MOPS, 1.0 g of SDS, and 0.3 g of EDTA into 1000 mL of deionized water. 10. 1-L baffled shake flasks. 11. Microcentrifuge tubes. 12. 0.2 μm sterile syringe filters. 13. Heating block for microcentrifuge tubes. 14. Incubator shaker. 15. Refrigerated high-speed centrifuge. 16. UV-Vis spectrophotometer.

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2.3 Purification of the UbAFCouK-sfGFP Probe

1. Deoxyribonuclease (DNase). 2. Lysozyme. 3. 200 mM PMSF stock solution: Dissolve 34.8 mg of phenylmethanesulfonyl fluoride (PMSF) in 1 mL of isopropanol; store at 4 °C. 4. Ni-NTA agarose. 5. Lysis buffer: 20 mM Tris–HCl, 500 mM NaCl, 10% glycerol, 10 mM imidazole, pH 8.0. 6. Elution buffer: 20 mM Tris–HCl, 500 mM NaCl, 10% glycerol, 250 mM imidazole, pH 8.0. 7. PBS buffer, pH 7.4. 8. Kit for determining protein concentration (e.g., BCA protein assay kit). 9. Ultrasonic cell disruptor. 10. Affinity chromatography columns. 11. Centrifugal filter units (e.g., Amicon Ultra-15 Centrifugal Filter Unit 10-kDa MWCO).

2.4 Analysis of Deubiquitinase Activities and Screening of Deubiquitinase Inhibitors Using the UbAFCouK-sfGFP Probe

1. USP7 (see Note 5). 2. Assay buffer: 20 mM Tris–HCl, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.4 (see Note 6). 3. Deubiquitinase candidate inhibitors: 10 mM stock solutions in DMSO. 4. 8-strip PCR tubes. 5. Multichannel pipettes. 6. 96-well optical-bottom plates. 7. Fluorescence microplate reader (e.g., Biotek Synergy H1).

3

Methods

3.1 Site-Specific Incorporation of AFCouK into Proteins in E. coli

1. Thaw a tube of BL21(DE3) competent E. coli cells (50 μL) on ice. 2. Add 30 ng of pACouKRS plasmid and 30 ng of pEGFPY39TAG or pUb-Y59TAG-sfGFP plasmid to the competent cells. Mix the cells and plasmids gently by pipetting up and down. Incubate the mixture on ice for 15 min. 3. Place the tube containing the mixture in a 42 °C heating block and incubate for 1 min. 4. Place the tube on ice for 2 min. 5. Add 900 μL of room temperature LB medium into the mixture and shake vigorously (e.g., 220 rpm) at 37 °C for 1 h.

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6. Centrifuge the tube for 1 min at 5000 g. 7. Remove 850 μL of the supernatant and resuspend the cell pellet in the remaining 100 μL of media by gentle pipetting. 8. Spread the mixture onto an LB agar plate to completely cover the plate and incubate overnight at 37 °C. 9. Pick a single colony from the agar plate and inoculate into 5 mL LB medium supplemented with 1000× kanamycin and chloramphenicol stock solutions (each 5 μL). Incubate the bacterial culture overnight at 37 °C in an incubator shaker at 220 rpm. 10. On the next day, use 5 mL of the overnight culture to inoculate 400 mL of LB supplemented with kanamycin and chloramphenicol in a baffled shake flask. 11. Incubate the bacterial culture at 37 °C in an incubator shaker at 250 rpm until the optical density at 600 nm reaches 0.6. 12. Once the optical density at 600 nm reaches 0.6, add 400 μL of 500 mM AFCouK into the bacterial culture. The final concentration of AFCouK should be 0.5 mM (see Note 7). 13. After 0.5 h additional incubation, add 400 μL of 1 M IPTG stock solution into the culture to induce the protein expression. Continue to incubate the bacterial culture at 37 °C in an incubator shaker at 220 rpm for 10 h. 14. Optional: Take an aliquot of the bacterial culture (e.g., 5 mL) and collect the cells by centrifugation at 8000 g for 30 min at 4 °C. Discard the supernatant and lyse the cells with SDS lysis buffer on a 95 °C heating block for 5 min. Clarify the resulting cell lysates with centrifugation at 16,000 g for 5 min. Analyze the samples by SDS-PAGE and western blot (see Note 8). 15. Harvest the cells by centrifugation at 8000 g for 30 min at 4 ° C. Discard the supernatant and freeze the cell pellet at -80 °C. 3.2 Purification of the UbAFCouK-sfGFP Probe

1. Thaw the cell pellets on ice and suspend in 10 mL of lysis buffer supplemented with 20 μg/mL DNase, 500 μg/mL lysozyme, and 75 μL of PMSF stock solution. Incubate the mixture at 4 ° C in an incubator shaker at 70 rpm for 1 h. For purification of the UbAFCouK-sfGFP probe, cells were co-transformed with the pACouKRS and pUb-Y59TAG-sfGFP plasmids. 2. Sonicate the mixture on ice with an ultrasonic cell disruptor using 3-s bursts at 30 W and 5-s cooling periods between each burst for a total of 15 min (see Note 9). 3. Spin down the mixture by centrifugation at 20,000 g for 30 min at 4 °C to pellet the cellular debris. 4. Pipet 1 mL of the 50% Ni-NTA slurry to a 15 mL tube and briefly centrifuge. Remove supernatant and wash the beads with 2 mL lysis buffer twice.

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5. Transfer the supernatant (from step 3 above) to the Ni-NTAcontaining 15 mL tube and mix gently by shaking in an incubator shaker at 70 rpm and 4 °C for 1 h. 6. Load the mixture into a column with the bottom outlet capped. 7. Remove the bottom cap of the column and collect the flowthrough. 8. Wash the Ni-NTA beads in the column with 10 mL of lysis buffer and elute the protein four times with 0.5 mL of elution buffer. Collect the wash fractions and eluate into individual tubes. 9. Analyze the protein purity in the flow-through, wash fractions, and eluate by SDS-PAGE. 10. Combine pure fractions and eluate into a centrifugal filter unit. 11. Cap the centrifugal filter unit and centrifuge the unit at 4000 g for 30 min at 4 °C. 12. Reconstitute the concentrate with PBS buffer to 4 mL and centrifuge the unit at 4000 g for 30 min at 4 °C. Repeat this buffer exchange process for three times to eliminate imidazole (see Note 10). 13. Collect and aliquot the concentrated protein. Measure the concentration using the BCA assay. Store the aliquots at 80 °C (see Note 11). 3.3 Fluorescence Detection of Deubiquitinase Activities Using the UbAFCouK-sfGFP Probe

1. Prepare a 320 nM USP7 solution in assay buffer (see Note 12). 2. Perform a series of twofold dilutions with assay buffer to prepare a range of concentrations (5–320 nM) of the USP7 solutions in 0.2 mL 8-strip PCR tubes. 3. Transfer 20 μL of the USP7 solutions (from step 2 above) with three replicates of each concentration to a 96-well opticalbottom black plate using a multichannel pipette. 4. Thaw the UbAFCouK-sfGFP protein probe on ice and dilute with assay buffer to a concentration of 0.625 μM. 5. Add 80 μL of the UbAFCouK-sfGFP probe solution to each well of the 96-well plate containing the USP7 solutions using a multichannel pipette. The final concentration of the UbAFCouK-sfGFP probe is 500 nM and final concentrations of USP7 are from 1 nM to 64 nM. 6. Monitor the 96-well plate on a fluorescence microplate reader set at 37 °C. Fluorescence intensities of each well are measured at 440 nm and 510 nm with excitation at 350 nm every 20 min over a period of 120 min (see Note 13) (Fig. 2a).

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Fig. 2 Analysis of deubiquitinase activities and screening of deubiquitinase inhibitors using the UbAFCouKsfGFP probe. (a) Fluorescence response kinetics of UbAFCouK-sfGFP towards USP7 at different concentrations. (b) Screening of candidate deubiquitinase inhibitors against USP7 with the UbAFCouK-sfGFP probe. Data are shown as mean inhibition rates from two replicates. (c) Dose-dependent inhibition against USP7 and IC50 analysis of the candidate USP7 inhibitor. Data are shown as mean ± s. d. (n = 3). (Reproduced from Ref. [19] with permission from the Royal Society of Chemistry) 3.4 Screening of Deubiquitinase Inhibitors Using the UbAFCouK-sfGFP Probe

1. Prepare the inhibitor solutions at two different concentrations (i.e., 10 and 100 μM) in assay buffer. Prepare the DMSO control solution by diluting an equal amount of DMSO with assay buffer (see Note 14). 2. Prepare a 100 nM USP7 solution in assay buffer (see Note 12). 3. Transfer 10 μL of the inhibitor solutions at different concentrations or the DMSO control solution with duplicates to 96-well optical-bottom black plates. 4. Add 10 μL of the USP7 solution (from step 2 above) or assay buffer to each well of the 96-well plates. 5. Incubate the mixture at 37 °C for 30 min. 6. Thaw the UbAFCouK-sfGFP protein probe on ice and dilute with assay buffer to a concentration of 0.625 μM. 7. Add 80 μL of the UbAFCouK-sfGFP probe solution to each well of the 96-well plates (from step 5 above) using a

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multichannel pipette. The final concentrations of the UbAFCouK-sfGFP probe and USP7 are 500 and 10 nM, respectively. The final concentrations of the inhibitors are 1 or 10 μM. 8. Incubate the plates at 37 °C for 120 min. 9. Read the 96-well plates on a fluorescence microplate reader. Fluorescence intensities of each well are measured at 440 and 510 nm with excitation at 350 nm. 10. Calculate the inhibition effect of every inhibitor by the following equation: Inhibition effect ð%Þ =

AC - X × 100% AC - NC

where “X” represents the fluorescence ratio at 440 nm and 510 nm (i.e., F440/F510) of the well in the presence of an inhibitor, “AC” is the fluorescence ratio (F440/F510) for the active control in the absence of an inhibitor (i.e., DMSO control solution + USP7 + UbAFCouK-sfGFP probe), and “NC” is the fluorescence ratio (F440/F510) for the negative control without USP7 (i.e., DMSO control solution + assay buffer + UbAFCouK-sfGFP probe) (see Note 15) (Fig. 2b). 3.5 IC50 Analysis of Deubiquitinase Inhibitors Using the UbAFCouK-sfGFP Probe

1. Prepare a 1 mM inhibitor solution in assay buffer. Prepare the DMSO control solution by diluting an equal amount of DMSO with assay buffer (see Note 14). 2. Perform a series of threefold dilutions with assay buffer to prepare a range of concentrations (from 0.152 μM to 1 μM) of the inhibitor solutions in 0.2 mL 8-strip PCR tubes. 3. Prepare a 100 nM USP7 solution in assay buffer (see Note 12). 4. Transfer 10 μL of the inhibitor solutions at different concentrations or the DMSO control solution with three replicates to 96-well optical-bottom black plates. 5. Add 10 μL of the USP7 solution (from step 3 above) or assay buffer to each well of the 96-well plates. 6. Incubate the mixture at 37 °C for 30 min. 7. Thaw the UbAFCouK-sfGFP protein probe on ice and dilute with assay buffer to a concentration of 0.625 μM. 8. Add 80 μL of the UbAFCouK-sfGFP probe solution to each well of the 96-well plates (from step 6 above) using a multichannel pipette. The final concentrations of the UbAFCouKsfGFP probe and USP7 are 500 and 10 nM, respectively. The final concentrations of the inhibitors are from 0.0152 μM to 100 μM. 9. Incubate the plates at 37 °C for 120 min.

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10. Read the 96-well plates on a fluorescence microplate reader. Fluorescence intensities of each well are measured at 440 and 510 nm with excitation at 350 nm. 11. Calculate the USP7 activity by the following equation: USP7 activity ð%Þ =

X - NC × 100% AC - NC

where “X” represents the fluorescence ratio at 440 and 510 nm (i.e., F440/F510) of the well in the presence of an inhibitor, “AC” is the fluorescence ratio (F440/F510) for the active control in the absence of an inhibitor (i.e., DMSO control solution + USP7 + UbAFCouK-sfGFP probe), and “NC” is the fluorescence ratio (F440/F510) for the negative control without USP7 (i.e., DMSO control solution + assay buffer + UbAFCouK-sfGFP probe) (see Note 16) (Fig. 2c).

4

Notes 1. Keep the AFCouK stock solution away from direct light. AFCouK can be obtained by custom synthesis following the reported protocol [19]. 2. The pACouKRS plasmid contains a copy of ACouKRS gene engineered from Methanosarcina mazei PylRS (Y306A/ L309A/348S/384F) under the control of the glnS promoter. The DNA sequence of ACouKRS is shown in Table 1. 3. The pEGFP-Y39TAG plasmid contains a C-terminally 6*Histagged EGFP-Y39TAG gene under the control of the bacteriophage T5 promoter and a Methanosarcina mazei tRNAPyl gene under the lpp promoter. The DNA sequences of EGFPY39TAG and tRNAPyl genes are shown in Table 1. 4. The pUb-Y59TAG-sfGFP plasmid contains a C-terminally 6*His-tagged ubiquitin-Y59TAG-sfGFP gene under the control of the bacteriophage T5 promoter and a Methanosarcina mazei tRNAPyl gene under the lpp promoter. The DNA sequences of ubiquitin-Y59TAG-sfGFP-21 and tRNAPyl genes are shown in Table 1. 5. USP7 is used as a representative deubiquitinase. Recombinant human USP7 catalytic domain (208–560) protein is commercially available. We purchased the recombinant human USP7 catalytic domain from Sino Biological (cat: 11681-HNCB). USP7 catalytic domain can also be recombinantly expressed and purified from E. coli [35]. 6. We recommend to use freshly prepared assay buffer since DTT is not stable in solution. 7. A control experiment can be performed in parallel without the addition of AFCouK.

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Table 1 DNA sequences used in this protocol Gene name

Sequence (5′ to 3′)

ACouKRS

atggataaaaagcctttgaacactctgatttctgcgaccggtctgtggatgtcccgcaccggcaccatccacaaaatcaaacac catgaagttagccgttccaaaatctacattgaaatggcttgcggcgatcacctggttgtcaacaactcccgttcttctcgtac cgctcgcgcactgcgccaccacaaatatcgcaaaacctgcaaacgttgccgtgttagcgatgaggacctgaacaaattcct gaccaaagctaacgaggatcagacctccgtaaaagtgaaggtagtaagcgctccgacccgtactaaaaaggctatgccaa aaagcgtggcccgtgccccgaaacctctggaaaacaccgaggcggctcaggctcaaccatccggttctaaattttctccg gcgatcccagtgtccacccaagaatctgtttccgtaccagcaagcgtgtctaccagcattagcagcatttctaccggtgcta ccgcttctgcgctggtaaaaggtaacactaacccgattactagcatgtctgcaccggtacaggcaagcgccccagctctga ctaaatcccagacggaccgtctggaggtgctgctgaacccaaaggatgaaatctctctgaacagcggcaagcctttccgt gagctggaaagcgagctgctgtctcgtcgtaaaaaggatctgcaacagatctacgctgaggaacgcgagaactatctgg gtaagctggagcgcgaaattactcgcttcttcgtggatcgcggtttcctggagatcaaatctccgattctgattccgctgg aatacattgaacgtatgggcatcgataatgataccgaactgtctaaacagatcttccgtgtggataaaaacttctgtctgcg tccgatgctggccccgaacctggctaactatgctcgtaaactggaccgtgccctgccggacccgatcaaaattttcgaga tcggtccttgctaccgtaaagagtccgacggtaaagagcacctggaagaattcaccatgctgaacttcagccagatggg tagcggttgcacgcgtgaaaacctggaatccattatcaccgacttcctgaatcacctgggtatcgatttcaaaattgttggt gacagctgtatggtgtttggcgatacgctggatgttatgcacggcgatctggagctgtcttccgcagtagtgggcccaat cccgctggatcgtgagtggggtatcgacaaaccttggatcggtgcgggttttggtctggagcgtctgctgaaagtaaaa cacgacttcaagaacatcaaacgtgctgcacgttccgagtcctattacaatggtatttctactaacctgtaa

EGFPY39TAG

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaag ttcagcgtgtccggcgagggcgagggcgatgccacctagggcaagctgaccctgaagttcatctgcaccaccggcaagc tgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagc agcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaag acccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacg gcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccc catcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgaga agcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagagatct catcaccatcaccatcactaa

tRNAPyl

tggcggaaaccccgggaatctaacccggctgaacggatttagagtccattcgatctacatgatcaggtttcc

Ub-Y59TAG- atgcagatcttcgtgaagaccctgaccggcaaaaccattaccctggaagtggaaccgagcgataccatcgagaacgtgaaag sfGFP ccaagatccaggacaaagaaggtattccgccggatcagcagcgcctgatctttgccggcaaacagctggaagatggtcg caccctgagcgattagaacatccagaaggagagcaccctgcatctggtgctgcgtctgcgcggtggtatagcatggcct ttgtccggactcagatccgctggcccagctggcccaggaggacgatcgggaatggttagcaaaggtgaagaactgttt accggcgttgtgccgattctggtggaactggatggtgatgtgaatggccataaatttagcgttcgtggcgaaggcgaa ggtgatgcgaccaacggtaaactgaccctgaaatttatttgcaccaccggtaaactgccggttccgtggccgaccctgg tgaccaccctgacctatggcgttcagtgctttagccgctatccggatcatatgaaacgccatgatttctttaaaagcgcga tgccggaaggctatgtgcaggaacgtaccattagcttcaaagatgatggcacctataaaacccgtgcggaagttaaattt gaaggcgataccctggtgaaccgcattgaactgaaaggtattgattttaaagaagatggcaacattctgggtcataaactg gaatataatttcaacagccataacgtgtatattaccgccgataaacagaaaaatggcatcaaagcgaactttaaaatccgtca caacgtggaagatggtagcgtgcagctggcggatcattatcagcagaataccccgattggtgatggcccggtgctgctg ccggataatcattatctgagcacccagagcgttctgagcaaagatccgaatgaaaaacgtgatcatatggtgctgctggaa tttgttaccgccgcgggcattacccacggtatggatgaactgtataaaggcagccaccatcatcatcaccattaa

8. Western blotting analysis using antibodies against the 6*His tag can be performed to examine the site-specific incorporation of AFCouK and expression of the AFCouK-containing proteins (e.g., EGFP-Y39AFCouK and Ub-Y59AFCouK-sfGFP). The full-length proteins are only expressed in the presence of AFCouK, but not in the absence of AFCouK.

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9. Keep the cells on ice during the whole process and make sure to let the mixture cool down between each sonication cycle. Sonication is used to disrupt cell membrane and extract the proteins from the pelleted cells. Avoid generating air bubbles in the sonication process. Air bubbles reduce the disruption efficiency and can be removed by brief centrifugation. 10. Keep the membrane in the centrifugal filter unit from drying out once wet. Collect concentrated proteins immediately after centrifugation for maximizing the sample recovery. 11. It is important to keep the protein samples at 4 °C during the purification and buffer exchange steps. The purified protein should be aliquoted and repeated freeze/thaw cycles should be avoided. The identity of the purified protein can be verified by mass spectrometry analysis. 12. USP7 is only utilized as an example. Other deubiquitinases with similar deubiquitinating activities can also be used. The USP7 stock solution should be aliquoted and stored at -80 °C. Avoid repeated freeze-thaw cycles since the activity of USP7 could be lost. 13. The fluorescence maxima of AFCouK and sfGFP are at 440 and 510 nm, respectively. The length and flexibility of the linker between Ub-Y59AFCouK and sfGFP can affect the fluorescence response of the probe towards USP7 [19]. 14. The DMSO control solution contains no inhibitor, which is used to assess the full activity of USP7. 15. The inhibition data can be processed and visualized as a heatmap. 16. The USP7 activity data were fitted with a four-parameter logistic model using GraphPad Prism 8.0 and IC50 values were calculated with this model.

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Chapter 5 Creating Selenocysteine-Specific Reporters Using Inteins Christina Z. Chung, Dieter So¨ll, and Natalie Krahn Abstract The selenium moiety in selenocysteine (Sec) imparts enhanced chemical properties to this amino acid and ultimately the protein in which it is inserted. These characteristics are attractive for designing highly active enzymes or extremely stable proteins and studying protein folding or electron transfer, to name a few. There are also 25 human selenoproteins, of which many are essential for our survival. The ability to create or study these selenoproteins is significantly hindered by the inability to easily produce them. Engineering translation has yielded simpler systems to facilitate site-specific insertion of Sec; however, Ser misincorporation remains problematic. Therefore, we have designed two Sec-specific reporters which promote highthroughput screening of Sec translation systems to overcome this barrier. This protocol outlines the workflow to engineer these Sec-specific reporters, with the application to any gene of interest and the ability to transfer this strategy to any organism. Key words Intein-based reporter, Selenoproteins, Synthetic biology, Genetic code expansion, Fluorescence

1

Introduction Selenocysteine (Sec), the 21st genetically encoded amino acid, is found in proteins in all domains of life. These proteins are termed selenoproteins and play essential roles in many organisms. Sec is unique in that it is biosynthesized on its cognate tRNA (tRNASec), being first aminoacylated by seryl-tRNA synthetase (SerRS). Following conversion to Sec, Sec-tRNASec is brought to the ribosome by a specialized elongation factor to decode UGA in the presence of an mRNA hairpin (Sec insertion sequence or SECIS). This generalized path for insertion of Sec involves additional proteins and features, which are not discussed, that differ amongst the three domains of life [1, 2]. The increased complexity of inserting Sec into proteins naturally, has limited the ability to overexpress selenoproteins and study the role of Sec in their essential functions. Over the past decade, new technologies have been developed to insert Sec via a simplified or rewired translation system [3–

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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6]. One strategy has been to use EF-Tu to recognize selenocysteinylated tRNA for insertion at a UAG codon, circumventing the need for a specialized elongation factor, the SECIS element, and other proteins required for Sec translation. However, since EF-Tu is specific for aminoacylated tRNA, it does not distinguish between Ser and Sec. This means that Ser can suppress a UAG codon in this engineered system if the conversion to Sec is not efficient. Therefore, to optimize the biosynthesis path to promote Sec insertion over misincorporation of Ser, reporters are needed which require the activity of the amino acid, rather than just suppression of the UAG codon. The benzyl viologen assay, which requires the presence of Sec in E. coli formate dehydrogenase H, has been a standard assay which has facilitated much of the work thus far [3, 4, 6]. However, it is time-consuming and cumbersome and requires the use of an anaerobic chamber. Since then, other reporter proteins with a catalytically active Cys were engineered to be active when Sec is inserted but not Ser. This takes advantage of the similar chemistries between Cys and Sec, for example, a disulfide-dependent β-lactamase [7], an autocatalytic phycobiliprotein (smURFP) [8], and most recently a DnaB mini-intein that can transform any reporter into a Sec-specific reporter [9]. The versatility of the Sec-specific DnaB mini-intein makes it attractive for the development of Sec reporters and subsequently Sec translation pathways in various expression systems. Inteins are enzymes which naturally participate in protein splicing of exteins (the protein fragments surrounding the intein) [10]. Intein splicing occurs in three main steps: (i) amide-thioester rearrangement of the first amino acid of the intein (position 1) and the amide bond which joins the intein and N-terminal extein, (ii) transesterification involving the first amino acid of the C-terminal extein (position +1), and (iii) asparagine cyclization involving the last amino acid of the intein (Asn) for its release. Finishing reactions then facilitate formation of an amide bond between the N- and C-terminal exteins [11]. The intein (DnaB M86 mini-intein) we have adapted has been previously evolved to be less dependent on the surrounding sequences to allow retained activity when placed into multiple genes [12–14]. We have shown that since DnaB M86 relies on a Cys at position 1 for efficient splicing activity, we can replace the Cys with Sec to probe for Sec insertion into a protein. Furthermore, we found the DnaB M86 mini-intein is inactive in the presence of Ser [9]. This means that since Ser can be misincorporated at UAG codons encoding for Sec, these reporters can distinguish between misincorporated amino acids and the presence of Sec (or Cys). Therefore, in the following sections we outline the strategy to engineer a Sec-specific reporter of your choice. We have already engineered a Sec-specific kanamycin resistance selective reporter (pAB_a02) [9] and a Sec-specific

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green fluorescent protein screening reporter (pB_04) [9] and have used the latter to explain the strategy and considerations when developing a new Sec-specific reporter.

2

Creating Sec-Specific Reporters Engineering strategies to create a simpler Sec insertion path for recombinant selenoprotein production require reporters. Because Sec is biosynthesized on its tRNA, there is an intermediate Ser-tRNASec that can also be inserted into the UAG codon designed for Sec. Therefore, it is important to not only have a reporter that recognizes insertion of an amino acid at a UAG codon, but also to specifically recognize the presence of Sec, distinguishing against its precursor Ser. This protocol describes how to design a Sec-specific reporter by inserting an intein into a reporter gene of interest. While this strategy can be used for any gene, we explain the steps of the process using sfGFP, a commonly used fluorescent reporter.

2.1

Equipment

1. Thermocycler. 2. Electrophoretic equipment to make and run agarose gels. 3. Gel imaging system (e.g., ChemiDoc) or UV transilluminator. 4. Razor blades. 5. 37
C incubator. 6. Spectrophotometer (e.g., NanoDrop). 7. Heat block. 8. Electroporator. 9. Microcentrifuge tube rotator (optional). 10. Shaking incubator. 11. Microcentrifuge. 12. Plate reader (e.g., BioTek Synergy HTX).

2.2 Reagents and Materials

1. Oligonucleotide primers (see Table 1). 2. High-fidelity DNA polymerase (e.g., PfuUltra II Fusion). 3. PCR buffer (e.g., 10x PfuUltra II buffer). 4. 10 mM dNTPs. 5. 0.2 mL PCR tubes. 6. 6 DNA loading dye. 7. 1% (w/v) agarose gel. 8. 1x TAE (Tris/acetate/EDTA) buffer: 40 mM Tris-base, 20 mM acetic acid, 1 mM EDTA. 9. DNA staining reagent (e.g., ethidium bromide or SYBR Safe).

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Table 1 Primers used for creating the sfGFP-intein variants Name

Sequence (50 -30 )

Notes

Pla-Fwd

GACGGATGGCCTTTTTGCG

Forward primer to linearize pBAD

Pla-rev

GTGACGCCGTGCAAATAATC

Reverse primer to linearize pBAD

Ins-Fwd

GTCCACATTGATTA TTTGCACGGCGTCACTCAC TGCCCGCTTTCC

Forward primer to amplify the sfGFP cassette from pET-15b and insert into pBAD

Ins-rev

GTTTGTAGAAACGCAAAAAGGCCA TCCGTCCAAAAAACCCC TCAAGACCCG

Reverse primer to amplify the sfGFP cassette from pET-15b and insert into pBAD

pB_sfGFP_28F

TCCGTACGCGGGGAAGGCGAAGG

Forward primer to linearize pB_sfGFP at position 28

pB_sfGFP_28R

GAATTTATGGCCGTTTACGTC

Reverse primer to linearize pB_sfGFP at position 28

M86_Cys_28F

GACGTAAACGGCCATAAATTC TGCATCTCCGGAGATAGTTTG

Forward primer to amplify M86(Cys) for insertion at position 28

M86_Cys_28R

CCTTCGCCTTCCCCGCGTACGGA GTTATGTACAATGATGTCA TTGGCG

Reverse primer to amplify M86(Cys) for insertion at position 28

Cys_to_Ser_28F

CCATAAATTCAGCATCTCCGGAGA TAGTTTG

Forward primer to mutate Cys to Ser for position 28 variant

Cys_to_Ser_28R

CTCCGGAGATGCTGAATTTA TGGCCGTTTAC

Reverse primer to mutate Cys to Ser for position 28 variant

pB_sfGFP_30F

TCCGGGGAAGGCGAAGGTGATGC

Forward primer to linearize pB_sfGFP at position 30

pB_sfGFP_30R

TACGGAGAATTTATGGCCGT

Reverse primer to linearize pB_sfGFP at position 30

M86_Cys_30F

ACGGCCATAAATTCTCCGTATGCA TCTCCGGAGATAGTTTG

Forward primer to amplify M86(Cys) for insertion at position 30

M86_Cys_30R

GCATCACCTTCGCCTTCCCCGGAG Reverse primer to amplify M86(Cys) for insertion at position 30 TTATGTACAATGATGTCA TTGGCG

Cys_to_Ser_30F

CATAAATTCTCCGTAAGCATC TCCGGAGATAGTTTG

Forward primer to mutate Cys to Ser for position 30 variant

Cys_to_Ser_30R

CTCCGGAGATGCTTACGGAGAA TTTATGGCC

Reverse primer to mutate Cys to Ser for position 30 variant

pB_sfGFP_72F

TCACGTTACCCAGATCATA TGAAGC

Forward primer to linearize pB_sfGFP at position 72

pB_sfGFP_72R

AAAGCACTGTACACCGTATG

Reverse primer to linearize pB_sfGFP at position 72 (continued)

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Table 1 (continued) Name

Sequence (50 -30 )

Notes

M86_Cys_72F

CATACGGTGTACAGTGCTTTTGCA TCTCCGGAGATAGTTTG

Forward primer to amplify M86(Cys) for insertion at position 72

M86_Cys_72R

GCTTCATATGATCTGGGTAACG TGAGTTATGTACAATGATGTCA TTGGCG

Reverse primer to amplify M86(Cys) for insertion at position 72

Cys_to_Ser_72F

GTACAGTGCTTTAGCATC TCCGGAGATAGTTTG

Forward primer to mutate Cys to Ser for position 72 variant

Cys_to_Ser_72R

CTCCGGAGATGCTAAAGCACTG TACACC

Reverse primer to mutate Cys to Ser for position 72 variant

pB_sfGFP_205F

AGCGTATTA TCAAAGGACCCAAACGAG

Forward primer to linearize pB_sfGFP at position 205

pB_sfGFP_205R

CTGAGTGGACAAGTAATGGTTG

Reverse primer to linearize pB_sfGFP at position 205

M86_Cys_205F

CAACCATTACTTGTCCACTCAG TGCATCTCCGGAGATAGTTTG

Forward primer to amplify M86(Cys) for insertion at position 205

M86_Cys_205R

CTCGTTTGGGTCCTTTGATAA TACGCTGTTATGTACAATGATG TCATTGGCG

Reverse primer to amplify M86(Cys) for insertion at position 205

Cys_to_Ser_205F

GTCCACTCAGAGCATCTCCGGAGA Forward primer to mutate Cys to Ser TAGTTTG for position 205 variant

Cys_to_Ser_205R

CTCCGGAGATGCTCTGAG TGGACAAGTAATG

Reverse primer to mutate Cys to Ser for position 205 variant

Cys_to_TAG_205F GTCCACTCAGTAGATCTCCGGAGA Forward primer to mutate Cys to TAG TAGTTTG for position 205 variant Cys_to_TAG_205R CTCCGGAGATCTACTGAG TGGACAAGTAATG

Reverse primer to mutate Cys to TAG for position 205 variant

Underlined sequences are complementary to the plasmid

10. DNA ladder (e.g., 1 kb, 2-log). 11. DpnI enzyme. 12. 1.5 mL Eppendorf tubes. 13. Gel purification kit. 14. NEBuilder HiFi DNA Assembly Master Mix. 15. Chemical or electrocompetent E. coli cloning cells (e.g., Top10, DH5α, Stellar). coli 16. Electrocompetent E. B-95.ΔAΔfabRΔselABC).

expression

17. Electroporation cuvettes (1 mm or 2 mm).

cells

(e.g.,

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18. Luria Broth (LB), autoclaved. 19. Antibiotic stocks, sterile filtered. 20. LB agar plates with appropriate antibiotics (100 mm-wide plates). 21. Culture tubes (e.g., 12 mL capped round-bottom culture tubes). 22. Plasmid miniprep kit. 23. PCR purification kit. 24. 24-well culture plates with lids. 25. 96-well black plates with clear bottoms. 26. Breathable sealing membrane (e.g., Breathe-Easy sealing membrane). 27. Plasmid of Sec insertion system (e.g., pSecUAG-Evol2 from Addgene #163148). 28. 20% (w/v) arabinose in ddH2O, sterile filtered (or other induction agent for the Sec insertion system). 29. 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) in ddH2O, sterile filtered (or other induction agent for the reporter). 30. 100 mM sodium selenite (Na2SeO3) solution in ddH2O, sterile filtered. 31. 20% (w/v) glucose in ddH2O, sterile filtered.

3

Methods This protocol covers the steps to convert a reporter protein to a Sec-specific reporter. We suggest using a reporter protein that has an established and reliable assay. These assays may require slight modifications to detect intein cleavage and protein splicing. The first section addresses points to consider when designing the reporter system. Using our previously created sfGFP-intein reporter [9] as an example, the remaining sections delve into details on how to convert it to a Sec-specific reporter. The strategies of cloning, mutagenesis, and screening can then be adapted to the reporter of interest.

3.1 Choosing a Reporter System

1. Choose a reporter to be engineered to be Sec-specific. This can be a selection reporter (e.g., conferring antibiotic resistance, providing an essential media component) or a screening reporter (e.g., luciferase, GFP). The reporter expression plasmid should be compatible with the plasmid for the Sec incorporation system (e.g., compatible replication of origins, different antibiotic resistance). If choosing an antibiotic

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selection reporter, ensure that the antibiotic resistance of the reporter, expression plasmid, and Sec incorporation system plasmid are all different. High copy number plasmids with origin of replications such as pBR322 and pMB1 are recommended [15]. However, this will likely have to be optimized together with the promoter (see Subheading 3.1, step 2) and type of reporter. 2. Determine the appropriate promoter for the reporter. We recommend using a constitutive promoter for selection reporters and an inducible promoter for screening reporters. Constitutive reporters allow a small buildup of the reporter enzyme prior to exposure of the antibiotic or to media lacking the essential component. This means that cells are given a chance to survive before their survival depends on the efficiency of inserting Sec into the intein. Selection for Sec-specific cell growth is measured at the end of an incubation period (e.g., overnight incubation). Screening reporters do not create this restriction on cell growth but rather rely on protein expression to produce a signal. Since the signal accumulates over time, a constitutive promoter would provide an advantage to Cys and Ser controls. This is because the reporter protein can be generated once the cells start growing, while the Sec system requires time to produce its own machinery before it can translate the UAG codon. The inducible promoter permits expression of the Sec machinery prior to reporter expression facilitating better comparison of the Sec results to the controls (see Note 1). 3. The M86 DnaB mini-intein has been evolved to be highly functional, regardless of the surrounding amino acid sequence [12]. However, splicing efficiency still varies, and multiple insertion sites should be tested [9, 12]. There are two major requirements for the insertion site: (i) to have the intein inserted immediately before a Ser residue to facilitate the DnaB intein splicing activity [12] and (ii) to insert the intein into an α-helix or β-sheet. Inserting the intein into the structured part of the reporter has a higher chance of disrupting the reporter function (see Note 2). This ensures that if the intein cannot cleave and splice, the reporter will not be functional [9]. A 3D structure of the reporter is helpful to choose these intein insertion sites, but secondary structure predictions are also helpful. 3.2 Cloning the Reporter Plasmid and Intein-Containing Variants

Once a reporter protein has been chosen, it should be cloned into an expression plasmid under an appropriate promoter. We originally chose pET-15b as the backbone for sfGFP (sfGFP with C-terminal His6-tag replaced the N-terminal His6-tag, thrombin site, and multiple cloning site) but were unable to get sufficient

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fluorescence. Therefore, we transferred it into a pBAD vector which has been previously successful in fluorescence assays [16]. However, because the tRNA-based Sec insertion systems used in the So¨ll lab (e.g., pSecUAG-Evol2) are under an arabinose promoter [3], we replaced the araBAD promoter with the T7 promoter, sfGFP gene, T7 terminator, and lacI sequences from pET-15b. This gave us the pB_sfGFP plasmid that we refer to as the wild-type (WT) reporter and will act as the backbone for intein insertions [9]. Details of this transformation are outlined below. 3.2.1 Transfer the T7sfGFP Cassette from pET15b to pBAD Vector

1. Design primers (Pla-Fwd and Pla-Rev, Table 1) to amplify pBAD around the araBAD promoter to remove it and linearize the vector for insertion of the sfGFP gene. These primers are completely complementary to the plasmid and do not have any overhangs (Fig. 1). They should be relatively short (10–20 nucleotides) and follow general primer design considerations (i.e., start and end with a C or G, approximately 50% GC, melting temperatures between 50
C and 60
C and within a few degrees of each other). 2. Design primers to amplify the cassette from pET-15b containing the T7 promoter, sfGFP gene, T7 terminator, and lacI (Ins-Fwd and Ins-Rev, Table 1). The 50 -end of these primers will be complementary to the primers used to amplify pBAD and the 30 -end will be complementary to the T7-sfGFP cassette. Ensure that the 50 -end of the forward primer is complementary to the reverse primer for the plasmid and the 50 -end of the reverse primer is complementary to the forward primer for the plasmid to ensure correct orientation of the gene (Fig. 1). The 50 part of these primers should be approximately 15–18 nucleotides in length and end with a G or C. The 30 -end of the primers should follow the general primer design considerations from Subheading 3.2.1, step 1 (see Note 3). 3. Amplify the pBAD vector and the T7-sfGFP cassette with a high-fidelity DNA polymerase (e.g., PfuUltra II Fusion). Refer to the manufacturer’s guidelines to prepare 25 μL PCRs and for details on the settings for the thermocycler (see Note 4). 4. Once the thermocycler protocol is complete, add 0.5 μL of DpnI to the vector PCR tube and mix by pipetting up and down. Incubate the reaction at 37
C (in an incubator or thermocycler) for 1 h to remove any remaining parent plasmid (see Note 5). 5. Add 5 μL of 6 DNA loading dye to the digested vector reaction and the T7-sfGFP cassette (insert) reaction, and load the entirety of both samples along with a DNA ladder onto a 1% (w/v) agarose gel containing a DNA staining reagent. Run the gel for the appropriate amount of time (e.g., 140 V for 20 min) in 1 TAE buffer.

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Fig. 1 Schematic of primer design and general cloning strategy. Primers are designed to linearize the expression plasmid through PCR (Pla-Fwd and Pla-Rev). The insert will be amplified using primers with 50 -overhangs that are complementary to the expression plasmid (Ins-Fwd and Ins-Rev). Once the PCR products have been purified, they will be ligated together using the NEBuilder HiFi DNA Assembly Master Mix

6. Image the gel using a UV transilluminator (stand-alone or part of a gel imaging system). Cut out the amplified vector and insert bands (using the DNA ladder to confirm the correct size) using a razor blade (see Notes 6 and 7), and place them into separate 1.5 mL Eppendorf tubes. 7. Purify the DNA from the excised gel piece using a gel purification kit. Measure the DNA concentration with a spectrophotometer. 8. Join the vector and insert together using the NEBuilder HiFi DNA Assembly Master Mix. Combine the vector and insert together in a 1:2 plasmid/insert ratio that contains 100 ng of plasmid DNA. Mix in an equal volume of the 2 NEBuilder HiFi enzyme. The reaction should be prepared in 0.2 mL PCR tubes and incubated at 50
C for 1 h using a thermocycler (or incubator) (see Note 8).

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9. After assembly, add the entire mixture to 50 μL of competent E. coli cloning cells (e.g., Top10, DH5α). These can be chemical or electrocompetent cells and can be self-made or bought from a company. For cells bought from a company, follow the manufacturer’s instructions for transformation. When using chemical competent cells, keep the DNA/cell mixture on ice for 30 min before heat-shock (30–45 s at 42
C followed by 2–5 min on ice). For electrocompetent cells, transfer the DNA/cell mixture to a cold sterile electroporation cuvette (1 mm or 2 mm) before shocking them at 1.8 V with an electroporator. 10. Add 1 mL LB media (or your bacterial cell culture media of choice) and incubate at 37
C on a rotator for 1 h. During the incubation period, prewarm (at 37
C) two LB agar plates containing the appropriate antibiotic concentration per transformation. 11. Plate 100 μL from the transformation onto one LB agar plate. To plate the entirety of the cells onto the remaining plate, centrifuge (30 s at 13,000  g) the remaining 900 μL of the transformation. Remove all but approximately 100 μL of the media, resuspend the cell pellet, and plate this onto the remaining LB agar plate. Leave the LB agar plates upside down in a 37
C incubator overnight (see Notes 9–11). 12. Take the LB agar plates out of the incubator the next morning, single colonies should be visible on them. Store the plates upside down at 4
C during the day. At mid–late afternoon, prepare three 5 mL precultures of LB media with the appropriate antibiotic concentration in culture tubes. Pick three individual colonies from the agar plates and resuspend each one in a preculture. Place the precultures in the shaking incubator overnight at 37
C. 13. The next morning, use a miniprep kit to isolate the plasmids from each of the precultures. We recommend making a glycerol stock of each preculture prior to collecting the cell pellet (a quick source for more plasmid DNA). These can be made by mixing 325 μL of the saturated preculture with 175 μL of 80% glycerol and storing the stock at 80
C. 14. Once the plasmids have been isolated, they should be sent for sequencing to a facility of your choice to verify that the T7-sfGFP cassette has been inserted into the pBAD plasmid and no mutations are present. The resulting plasmid is referred to as pB_sfGFP [9]. Follow the facility’s instructions for submitting samples (see Note 12).

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Fig. 2 Crystal structure of sfGFP (PDB: 2B3P) showing the possible intein insertion sites. Highlighted in burgundy and annotated with black arrows (S, Ser; R, Arg) 3.2.2 Identify Sites of Insertion for the M86 DnaB Mini-Intein

For sfGFP, the insertion sites will be determined using a PDB structure (PDB ID: 2B3P, Fig. 2) and the UniProt entry of GFP from Aequorea victoria (P42212). Although the UniProt entry is not of sfGFP, it is the original GFP molecule and a supporting tool to decide where to insert the intein (see Note 13). 1. Identify Ser residues within a secondary structure element to insert the M86 DnaB mini-intein [12]. Based on the crystal structure, positions 28, 72, 86, 99, 202, 205, and 208 were identified as potential candidates. The UniProt entry agreed with all but position 99 and suggested two new sites at position 30 and 147. To manage the workload, a select number of sites were chosen from these: positions 28, 30, 72, and 205. These sites are spaced out within the protein; positions 28 and 205 are within a β-sheet, while 72 is near the active site. In the sfGFP sequence we used, position 30 is an Arg, not Ser. However, it is located in the middle of a β-sheet, a good candidate to disrupt the fluorescence, so by mutating Arg30Ser (see details in Subheading 3.2.2, step 2), this position could be used for intein insertion (see Note 2). 2. To insert the Cys-containing M86 DnaB mini-intein at the four chosen sites, primers were designed in a similar fashion as Subheading 3.2.1, step 1 and Subheading 3.2.1, step 2. To linearize the plasmid, the forward primer (pB_sfGFP_28F, pB_sfGFP_72F, pB_sfGFP_205F; Table 1) is complementary to the Ser codon and the downstream nucleotides. For position 30, the forward primer (pB_sfGFP_30F; Table 1) will be complementary to the codon at position 31 and the downstream

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nucleotides with the addition of three nucleotides (the Ser codon AGC) to the 50 -end of the primer. The reverse primer will be complementary to the nucleotides immediately upstream of the Ser codon (pB_sfGFP_28R, pB_sfGFP-30R, pB_sfGFP_72R, pB_sfGFP_205R; Table 1). The steps in Subheading 3.2.1, step 2 can be followed to design the primers (M86_Cys; Table 1) to amplify the intein from its original plasmid [12]. 3. For the cloning and to verify intein insertion, follow Subheading 3.2.1, steps 3–14. 3.2.3 Create Cys-to-Ser Substitution

After obtaining successful pB_sfGFP-intein plasmids with Cys at position 1, the next step is to mutate the Cys residue to Ser. The Ser-containing intein variant will act as a negative control since the M86 DnaB mini-intein should be inactive with Ser in position 1. These negative controls are important because tRNASec is initially charged with Ser and may be inserted into the growing polypeptide chain instead of Sec (see Note 14). 1. Design the primers to mutate Cys to Ser (AGC). The forward (Cys_to_Ser_F; Table 1) and reverse (Cys_to_Ser_R; Table 1) primers are advised not to be perfectly complementary to each other to reduce competition with primer dimers in the PCR. The primers should include about 10 nucleotides upstream and about 15–18 nucleotides downstream of the mutation site (see Note 15). Following the general primer design guidelines, it is important to have a C or G at both ends of the primers. 2. Amplify the plasmid with the correct primer set as in Subheading 3.2.1, step 3 with an extension time of 1 min/kb. Mix 5 μL of the reaction with 1 μL of 6 DNA loading dye and load the samples along with a DNA ladder onto a 1% (w/v) agarose gel and run for the appropriate amount of time (e.g., 140 V for 20 min). Image the gel with a gel imaging system to ensure the correct sized band is present. Follow Subheading 3.2.1, step 4 to digest any remaining parent plasmid. 3. Optional: Clean up the digested product with a PCR cleanup kit; follow the manufacturer’s protocol. Measure the DNA concentration with a spectrophotometer (e.g., NanoDrop) or other DNA measuring technique. 4. Transform 100 ng of the PCR cleaned up reaction or 5 μL of the DpnI-digested reaction. Follow details in Subheading 3.2.1, steps 9–14 for E. coli transformation and methods to verify the mutation.

3.3 Developing the Intein Assay with Cys and Ser Variants

To choose the best position for the intein, you need to screen the Cys- and Ser-containing intein variants. With sfGFP we monitor a fluorescence signal which should only be observed with the

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Fig. 3 Details on sfGFP-intein assay. (a) Schematic of the general sfGFP-intein assay. Briefly, E. coli cells are transformed with both the Sec expression plasmid and sfGFP-intein reporter. After an overnight incubation at 37
C, single colonies are transferred to a well of a 24-well plate to grow at 37
C for 6–8 h. The cultures are transferred again to a 96-well plate (Rep, replicate; V, variant) and fluorescence is measured in a plate reader in the presence and absence of an inducing agent for the expression of the sfGFP-intein reporter. (b) Results of the sfGFP-intein reporter where the intein was inserted into various positions of sfGFP to compare Cys and Ser insertion. (c) The final reporter with the intein inserted at position 205 was tested in the presence and absence of pSecUAG-Evol2. Position 1 of the intein encodes for either Cys, Ser, Gly, or Sec

Cys-containing variants and not the Ser-containing variants. Comparing the fluorescence signals to the WT signal informs you of the intein’s influence on sfGFP activity (Fig. 3). The sfGFP reporter is under an inducible promoter; therefore, the assay was designed to mimic standard recombinant protein expression. 1. Transform 100 ng of reporter DNA (cloned in Subheading 3.2) and 100 ng of the Sec system expression plasmid (e.g., pSecUAG-Evol2) into electrocompetent expression cells (e.g., B-95.ΔAΔfabRΔselABC). Transformations will need to

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be performed with the WT reporter and each of the Cys- and Ser-containing variants. Follow Subheading 3.2.1, steps 9–11 for the transformation and cell plating guidelines (see Note 16). 2. The next morning, prepare 24-well culture dishes with 0.5 mL media in each well containing the appropriate antibiotics for the two plasmids, any inducing agent for the Sec system (e.g., 0.1% [w/v] arabinose for pSecUAG-Evol2), selenium source (e.g., 10 μM sodium selenite), and 1% (w/v) glucose. The glucose is used to suppress leaky expression from the T7 promoter to promote consistent and clean results when comparing between induced and uninduced cultures. You will need four wells per reporter variant for the biological replicates. 3. Pick a colony for each of the four wells from a single reporter variant and resuspend in the media. Place lids on the plate (s) and incubate in a shaking incubator at 37
C for about 6–8 h (to A600 around 0.6) (see Note 17). 4. Prepare a 96-well plate to induce the cultures in the 24-well culture plates (Fig. 3a). There will be two media conditions on the plate: one with IPTG and one without IPTG. Rows A–D will contain IPTG and rows E–H will not have IPTG. Each column will correspond to a reporter variant (e.g., column 1 is WT, column 2 is Cys variant for position 28) and each row will be a biological replicate, with each replicate present in each media condition (e.g., rows A and E will have biological replicate 1; rows B and F will have biological replicate 2). 5. Pipette 75 μL of media containing the appropriate antibiotics for the two plasmids, any inducing agent for the Sec system (e.g., 0.1% [w/v] arabinose for pSecUAG-Evol2), selenium source (e.g., 10 μM sodium selenite), 1% (w/v) glucose, and 2 mM IPTG (to induce the reporter plasmid) into wells in rows A–D and 75 μL of media containing the appropriate antibiotics for the two plasmids, any inducing agent for the Sec system (e.g., 0.1% [w/v] arabinose for pSecUAG-Evol2), selenium source (e.g., 10 μM sodium selenite), and 1% (w/v) glucose into wells in rows E–H. 6. Transfer 75 μL from the 24-well culture plates into the appropriate wells in the 96-well plate (see Note 18). 7. Cover the plate with a breathable sealing membrane and place it on a plate reader (e.g., BioTek Synergy HTX). The plate reader should be set to incubate the plate at 37
C and take fluorescence (e.g., Ex. 485 nm, Em. 528 nm) and A600 readings every 15 min for 24 h. Additionally, the plate should be shaking between readings to facilitate cell growth. 8. Analyze the data to find the insertion site that gives the highest signal with the Cys-containing intein variant and the lowest

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signal with the Ser-containing intein variant. Data at various timepoints can be analyzed (e.g., 8, 12, and 16 h) by dividing the fluorescence readings by the A600 and taking the average of the four biological replicates for the individual variant with and without IPTG. The value of the uninduced condition (i.e., no IPTG) is subtracted from the induced condition to take the background fluorescence into consideration. The values and error bars can be represented in a bar graph for better comparison (Fig. 3b). Based on these results, position 205 (pB_sfGFP_205) was chosen as the most sensitive position for intein cleavage and discrimination between Cys and Ser. In addition, the Cys-containing variant produced the highest signal which is ideal when switching to a Sec-containing intein. Suppression and Sec incorporation are much slower and will lead to a lower signal than what is seen for Cys. 3.4 Test the SecIntein Assay for Its Specificity

1. Once a position has been chosen with the Cys- and Ser-containing intein variants, a Sec-containing intein variant must be prepared. 2. Mutate the Cys or Ser codon from their respective plasmids to TAG to encode for Sec. This can be done following Subheading 3.2.3, steps 1–4 using primers Cys_to_TAG_205F and Cys_to_TAG_205R (Table 1) (see Note 19). 3. Once all three intein variants (Cys, Ser, Sec) have been generated, it is time to test the sensitivity of the reporter with a Sec incorporation system. In this example, we will continue with pSecUAG-Evol2. Subheading 3.3, steps 1–8 were repeated with transformations containing pSecUAG-Evol2 and the reporter variants (e.g., pB_sfGFP_205C, pB_sfGFP_205S, pB_sfGFP_205U) or just the reporter variant (Fig. 3c). In this experiment, signals should only be observed for the Cys-containing variant (pB_sfGFP_205C) in the presence and absence of pSecUAG-Evol2 and the Sec-containing variant (pB_sfGFP_205U) only in the presence of pSecUAG-Evol2. No signal should be observed with the Sec-containing variant (pB_sfGFP_205U) in the absence of pSecUAG-Evol2 and the Ser-containing variant (pB_sfGFP_205S) in the presence and absence of pSecUAG-Evol2. These results inform us that the reporter is active with Sec but not Ser and only in the presence of the Sec incorporation system (i.e., no cross-reactivity with any endogenous cellular components). 4. Once the intein reporter (pB_sfGFP_205U) has been verified to be Sec-specific, it can be used to screen different Sec incorporation systems prior to more tedious Sec verification assays such as protein expression and purification.

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Notes 1. Choosing the vector and promoter for these assays can have a big impact on the sensitivity and reliability of the results obtained. We have outlined what worked best in our hands, but depending on the reporter of interest, alterations to the suggestions may need to be made. Testing different systems with the Cys-containing intein is advised before continuing with mutagenesis to Ser and Sec. 2. If there are no Ser residues present in structured regions of the protein, then a Ser can be inserted in place of Thr or Ala, as long as the mutation does not affect the reporter function. This can be tested through an activity assay. 3. The melting temperature should be determined for the annealed region at the 30 -end of the primers. The nucleotides on the 50 -extension do not anneal to the template and therefore should not be included when calculating the melting temperature. 4. Amplification of large vectors (~9 kbp or larger) may require alternative reaction conditions (e.g., additional dNTPS, reduced extension temperature, longer extension time). 5. Up to 2 μL of DpnI can be added to the PCR (1/10 the reaction volume) or overnight incubation at 37
C to ensure complete digestion of the parent plasmid. 6. Good PCR amplification will result in a clean single band. However, sometimes if annealing temperatures are low in the PCR protocol, this can lead to nonspecific binding and additional products. Regardless of how the gel looks, if there is a band at the appropriate size, it is generally safe to assume that this is the product of interest and cutting it out will separate it from everything else. Alternatively, the PCR protocol can be optimized to result in one band. 7. Limit the exposure time of the gel to UV irradiation. Extended periods of UV exposure can lead to random mutations in the DNA fragment, preventing successful sequencing later. 8. The vector/insert ratio can be adjusted to promote efficient assembly. Ratios of 1:3 or 1:5 are also commonly used. 9. Recovery of cells in Super Optimal broth with Catabolite repression (SOC) instead of LB can promote colony formation on the plates. 10. Plates should be placed into the incubator mid–late afternoon to avoid overgrowth of cells.

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11. Alternatively, one plate can be used per transformation, using one-half of the plate for the first 100 μL and the other half for the spun-down cell pellet. 12. If sequencing was not successful at identifying the gene insertion, additional colonies may need to be picked and the DNA isolated again. 13. Use any resources that are available to choose sites in the reporter which will disrupt function but also facilitate intein cleavage (i.e., presence of a Ser). 14. Another negative control that can be made is Gly. A Gly-containing intein reporter will be catalytically inactive and can therefore be compared to the Ser-containing intein reporter to ensure any output is only background. 15. The length of these primers will be reliant on the need to start and end with a G or C, while maintaining a melting temperature of at least 55
C. Because primer dimers can still occur, it is best to make slightly higher melting temperature primers and choose a higher annealing temperature for the reaction (e.g., 63
C). 16. Although there is no need for the tRNASec insertion machinery to obtain intein splicing and detection of fluorescence, the addition of this plasmid will affect cell growth and should therefore be included during assay development. 17. You can check the A600 value by using a plate reader. Alternatively, if you only have one 24-well plate, you can monitor cell growth throughout the day and move to Subheading 3.3, step 4 when the cells are ready to be induced. 18. Since there was no IPTG in the culture from the 24-well plate, transferring 75 μL of culture to the 75 μL of media will dilute the 2 mM IPTG to 1 mM (an appropriate concentration for induction). 19. Multiple intein positions can be chosen for mutagenesis to TAG if desired.

Acknowledgements We thank Oscar Vargas-Rodriguez and Kexin Meng for experimental advice and helpful discussions. This work was supported by grants from the National Institute of General Medical Sciences (R35GM122560-05S1 to D.S.) and, for the genetic studies, the Department of Energy Office of Basic Energy Sciences (DE-FG0298ER2031 to D.S.). Christina Z. Chung holds a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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References 1. Meng K, Chung CZ, So¨ll D, Krahn N (2022) Unconventional genetic code systems in archaea. Front Microbiol 13:1007832. https://doi.org/10.3389/fmicb.2022. 1007832 2. Chung CZ, Krahn N (2022) The selenocysteine toolbox: a guide to studying the 21st amino acid. Arch Biochem Biophys 730: 109421. https://doi.org/10.1016/j.abb. 2022.109421 3. Mukai T, Sevostyanova A, Suzuki T, Fu X, So¨ll D (2018) A facile method for producing selenocysteine-containing proteins. Angew Chem Int Ed Engl 57(24):7215–7219. https://doi.org/10.1002/anie.201713215 4. Aldag C, Bro¨cker MJ, Hohn MJ, Prat L, Hammond G, Plummer A, So¨ll D (2013) Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation. Angew Chem Int Ed Engl 52(5):1441–1445. https://doi.org/10.1002/anie.201207567 5. Thyer R, Robotham SA, Brodbelt JS, Ellington AD (2015) Evolving tRNASec for efficient canonical incorporation of selenocysteine. J Am Chem Soc 137(1):46–49. https://doi. org/10.1021/ja510695g 6. Miller C, Bro¨cker MJ, Prat L, Ip K, Chirathivat N, Feiock A, Veszpremi M, So¨ll D (2015) A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro. FEBS Lett 589(17):2194–2199. https://doi.org/10.1016/j.febslet.2015. 06.039 7. Majiduddin FK, Palzkill T (2003) Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics. Antimicrob Agents Chemother 47(3):1062–1067. h t t p s : // d o i . o r g / 1 0 . 1 1 2 8 / A A C . 4 7 . 3 . 1062-1067.2003 8. Thyer R, Shroff R, Klein DR, d’Oelsnitz S, Cotham VC, Byrom M, Brodbelt JS, Ellington AD (2018) Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains. Nat Biotechnol 36(7):

624–631. https://doi.org/10.1038/nbt. 4154 9. Chung CZ, Krahn N, Crnkovic´ A, So¨ll D (2022) Intein-based design expands diversity of selenocysteine reporters. J Mol Biol 434(8):167199. https://doi.org/10.1016/j. jmb.2021.167199 10. Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5(1): 4 4 6 – 4 6 1 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 9 / C3SC52951G 11. Mills KV, Johnson MA, Perler FB (2014) Protein splicing: how inteins escape from precursor proteins. J Biol Chem 289(21):14498–14505. https://doi.org/10.1074/jbc.R113.540310 12. Appleby-Tagoe JH, Thiel IV, Wang Y, Wang Y, Mootz HD, Liu XQ (2011) Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. J Biol Chem 286(39):34440–34447. https://doi. org/10.1074/jbc.M111.277350 13. Stevens AJ, Sekar G, Shah NH, Mostafavi AZ, Cowburn D, Muir TW (2017) A promiscuous split intein with expanded protein engineering applications. Proc Natl Acad Sci U S A 114(32):8538–8543. https://doi.org/10. 1073/pnas.1701083114 14. Marshall CJ, Grosskopf VA, Moehling TJ, Tillotson BJ, Wiepz GJ, Abbott NL, Raines RT, Shusta EV (2015) An evolved Mxe GyrA intein for enhanced production of fusion proteins. ACS Chem Biol 10(2):527–538. https://doi. org/10.1021/cb500689g 15. Phillips GJ, Park SK, Huber D (2000) High copy number plasmids compatible with commonly used cloning vectors. BioTechniques 28(3):400–408. https://doi.org/10.2144/ 00283bm02 16. Tharp JM, Ad O, Amikura K, Ward FR, Garcia EM, Cate JHD, Schepartz A, So¨ll D (2020) Initiation of protein synthesis with non-canonical amino acids in vivo. Angew Chem Int Ed Engl 59(8):3122–3126. https://doi.org/10.1002/anie.201914671

Chapter 6 Protein Expression with Biosynthesized Noncanonical Amino Acids Yong Wang, Wenkang Cai, Boyang Han, and Tao Liu Abstract Natural proteins are normally made by 20 canonical amino acids. Genetic code expansion (GCE) enables incorporation of diverse chemically synthesized noncanonical amino acids (ncAAs) by orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs using nonsense codons, which could significantly expand new functionalities of proteins in both scientific and biomedical applications. Here, by hijacking the cysteine biosynthetic enzymes, we describe a method combining amino acid biosynthesis and GCE to introduce around 50 structurally novel ncAAs into proteins by supplementation of commercially available aromatic thiol precursors, thus eliminating the need to chemically synthesize these ncAAs. A screening method is also provided for improving the incorporation efficiency of a particular ncAA. Furthermore, we demonstrate bioorthogonal groups, such as azide and ketone, that are compatible with our system and can be easily introduced into protein for subsequent site-specific labeling. Key words Genetic code expansion, Non-canonical amino acids, Biosynthesis, Aromatic thiol precursors, Bioorthogonal conjugation

1

Introduction Most natural proteins consist of 20 canonical amino acids and can be subjected to different post-translation modifications (PTMs), such as methylation, acetylation, and glycosylation, conferring structural and functional diversity to proteins [1, 2]. However, driven by the demands of scientific and biomedical applications, the limited diversity of canonical amino acids has prompted the development of proteins with diverse noncanonical amino acids (ncAAs). In this regard, genetic code expansion (GCE), pioneered by Schultz and coworkers [3], is one of the most commonly used strategy, and the number of genetically incorporable ncAAs is over 200. Specifically, GCE employs engineered orthogonal

Authors Yong Wang and Wenkang Cai have contributed equally to this work. Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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aminoacyl-tRNA synthetase (aaRS)/tRNA pairs for incorporation of ncAAs and has great potential for both therapeutic and biotechnological applications, including modulation of enzyme activity [4], spectroscopy [5] or imaging probes [6], bioorthogonal conjugation with other tags (e.g., fluorophores, polyethylene glycol, drugs, radioactive isotopes, immunotoxins, nucleic acids, etc.) [7– 11], simulation of PTM [12], and so on. Although GCE is considered nowadays a well-established strategy in protein engineering, its potentials have not been fully exploited. The ncAAs must be exogenously fed to cells, typically at concentrations of a few mM at multiliter scale for recombinant protein expression. Thus, broad application of this technique is hampered by the limited commercial availability and high cost of ncAAs as chemical preparation of these amino acids often involve multiple synthetic and purification steps. Inspired by pyrrolysine, a ncAA biosynthesized from lysine in nature [13], there are several strategies to produce ncAAs in situ from inexpensive and commercially available media supplements in E. coli with certain biosynthetic pathway. This offers a promising way to reduce the cost and synthetic burden to obtain these ncAAs. The resulting ncAAs can be subsequently incorporated into proteins by GCE. For example, as early as 2003, Mehl and Schultz demonstrated biosynthesis of p-aminophenylalanine and its genetic incorporation into proteins in E. coli [14]. Xiao et al. optimized this system to allow functionalization of the resulting proteins by chemical conjugation reactions [15]. Recently, Xiao et al. also developed an autonomous system to produce 5-hydroxytryptophan with a unique chemical functionality for site-specific protein modification after introducing by GCE [16]. Lee et al. accomplished five Phe and Tyr derivatives from α-keto acid precursors by introducing glutamine-phenylpyruvate aminotransferase into E. coli [17]. In another example, they also developed the biosynthetic pathway of L-dihydroxyphenylalanine from catechol and then incorporated into specific recombinant proteins [18]. Similarly, Budisa et al. reported the biosynthesis of S-allylcysteine in situ and then introduced into proteins for further bioorthogonal conjugation [19]. In 2017, the Chin group demonstrated biosynthesis of phosphothreonine and its incorporation into proteins by pThrRS–tRNAv2.0CUA pair in cells with GCE, solving the problem of cell impermeability of phosphothreonine [20]. However, there are two main limitations in the above examples preventing their wider application. Firstly, most examples only demonstrated biosynthesis of only one ncAA at a time. Secondly, expression yields of protein variants containing the biosynthesized ncAAs were rather low. Our group is interested in developing a more general and highly efficient system for expression of recombinant proteins containing customized ncAAs, biosynthesized from economical commercially available precursors with diverse

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Fig. 1 Recombinant expression of proteins containing noncanonical amino acids biosynthesized from aromatic thiols

side-chain structures and functionalities for desired applications and for convenient screening of proteins with altered function. Inspired by Maier et al. hijacking the cysteine biosynthetic pathway to produce ncAAs through semisynthesis [21], herein, we describe a protocol for reengineering the cysteine biosynthetic pathways and screening aromatic thiol anylogues as highly nucleophilic cores to efficiently trap cysteine biosynthetic intermediates to produce ncAAs under physiological conditions and for reactions of biosynthesized ncAAs containing bioorthogonal groups incorporated into proteins by means of the MjTyrosyl GCE system with hydroxyamine- and DBCO-functionalized labels [22] (Fig. 1).

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2.1 Aromatic Thiol Precursors

1-(4-Mercaptophenyl)ethan-1-one and 4-azidobenzenethiol are obtained by chemical synthesises. Other aromatic thiol precursors are commercially available. 1. 1-(4-Iodophenyl)ethan-1-one. 2. Copper sulfate pentahydrate. 3. Cesium carbonate. 4. Dimethyl sulfoxide. 5. Inert gas (e.g., argon). 6. Deionized water. 7. 5% HCl(aq): Add 13.5 g concentrated HCl into 86.5 g H2O. 8. Petroleum ether (PE). 9. Ethyl acetate (EA). 10. Rotary evaporator. 11. Anhydrous magnesium sulfate. 12. 200–300 mesh silica gel.

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13. Chromatography column. 14. Acetonitrile. 15. Tert-butyl nitrite (t-BuONO). 16. 4-Aminothiol. 17. Trimethylsilyl azide. 18. Desiccator. 2.2 Protein Expression in E. coli

1. E. coli DH10B chemical competent cells. 2. E. coli BL21 (DE3) electrocompetent cells. 3. Plasmid pET22b with the gene of interest containing an amber codon (TAG) at the site for ncAA (see Note 1). 4. Plasmid pBK-CysM-NtSat4 (see Note 2). 5. Plasmid pUltra-CNF (available through Addgene: plasmid# 48215). 6. Plasmid pUltra-PhSeRS/tRNAtyr (see Note 3). 7. 2YT medium: 12.0 g/L yeast extract, 16.0 g/L tryptone, and 5.0 g/L NaCl. 8. LB agar: 5.0 g/L yeast extract, 10.0 g/L tryptone, 10.0 g/L NaCl, and 15.0 g/L agar. 9. PCR polymerase (e.g., KOD One TM PCR Master Mix from TOYOBO). 10. Restriction enzyme DpnI. 11. 100 mg/mL ampicillin stock solution. 12. 50 mg/mL spectinomycin stock solution. 13. 50 mg/mL kanamycin stock solution. 14. 10
phosphate-buffered saline (PBS, pH 7.4; see Note 4). 15. 1 M IPTG: Dissolve 0.238 g isopropyl-β-D-thiogalactopyranoside in 1 mL water. 16. Gibson Assembly Master Mix (NEB, cat# E2611L). 17. Plasmid extraction kit.

2.3 Protein Purification

1. Binding buffer: 20 mM Tris–HCl, 500 mM NaCl, 10 mM imidazole, pH 8.0 (see Note 4). 2. Wash buffer: 20 mM Tris–HCl, 500 mM NaCl, 25–50 mM imidazole, pH 8.0 (see Note 4). 3. Elution buffer: 20 mM Tris–HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0 (see Note 4). 4. Lysis buffer: 20 mM Tris–HCl, 500 mM NaCl, 10 mM imidazole, pH 8.0, 1 mg/ml lysozyme. 5. Amicon Ultra column (Millipore).

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Fig. 2 Structure of DBCO-mPEG20K (a), diSulfo CY5 DBCO (methyl) (b), and hydroxyamine-mPEG20K (c)

6. Ni-NTA agarose. 7. Lysozyme. 2.4 Protein SiteSpecific Modification

1. DBCO-mPEG20K Fig. 2a).

(Confluore,

Cat#

BCD-74-50

mg,

2. DiSulfo CY5 DBCO (methyl) (hereinafter referred to DBCOCY5, Confluore, cat# BDC-25-25 mg, Fig. 2b). 3. Hydroxyamine-mPEG20K (custom synthesis from Confluore, Fig. 2c). 4. Kit for determination of protein concentration (e.g., Pierce BCA protein assay kit). 5. Size-exclusion column for protein purification (e.g., Cytiva Superdex 75 Increase 10/300 GL column). 6. Conjugation buffer: 6.5 mM KH2PO4, 95.0 mM K2HPO4, 125.0 mM NaCl, pH 8.4. 2.5

Selection

1. Plasmids: pBK-PhSeRS, pRep, pNeg. 2. E. coli DH10B electrocompetent cells. 3. DNA gel extraction kit. 4. GMML plate (M9 minimal medium supplemented with 1% glycerol, 300 μM leucine, 1 mM MgSO4, 0.1 mM CaCl2, 0.5% NaCl, 1000
heavy metal salts, 1 mg/L D-biotin, 1 mg/L thiamine–HCl, and 15 mg/L agar.) 5. 20% (w/w) arabinose. 6. 10 mg/mL tetracycline stock solution. 7. 68 mg/mL chloramphenicol stock solution.

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Fig. 3 Synthetic scheme of 1-(4-mercaptophenyl)ethan-1-one (a) and 4-azidobenzenethiol (b)

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Methods

3.1 Synthesis of Thiol Precursors Containing Keto (Fig. 3a) or Azide Group (Fig. 3b) 3.1.1 Synthesis of 1-(4Mercaptophenyl)Ethan-1One

1. Oven-dry a 25 mL double-neck round-bottom flask and magnetic rotor, and then cool slowly to room temperature in a desiccator over anhydrous calcium sulfate. 2. Add 1-(4-iodophenyl) ethan-1-one (0.25 g, 1.00 mmol), CuSO45H2O (12.50 mg, 0.05 mmol), Cs2CO3 (1.62 g, 5.00 mmol), DMSO (2.00 mL) to the round-bottom flask. 3. Place a reflux condenser (with flowing cooling water) on top of the double-neck round-bottom flask. 4. Fill the flask with the inert gas and seal the reaction flask. 5. Add 1,2-ethanedithiol (0.18 mL, 2.00 mmol) via a 1 mL syringe and stir the reaction mixture at room temperature, and then heat up to 110  C for 20 h using an oil bath. 6. After being cooled to ambient temperature, distribute the mixture in 100 mL of 5% HCl(aq) and 100 mL of ethyl acetate to a separatory funnel. Shake vigorously to mix the two phases, and collect the organic layer. Repeat the process with 50 mL of ethyl acetate for three times. 7. Wash the combined organic phases with brine (50 mL
3), and dry over anhydrous MgSO4. 8. Filter off the MgSO4, and concentrate the organic mixture by rotary evaporation. 9. The crude product is further purified by silica gel column chromatography using 25% EA in PE (see Note 5) to afford the 1-(4-mercaptophenyl)ethan-1-one as a yellow oil (100 mg, 65.00% yield) [23]. ESI-()-MS (M-H) is calculated for C8H8OS: 151.03; found: 151.03.

3.1.2 Synthesis of 4Azidobenzenethiol

1. Oven-dry a 25 mL single-neck round-bottom flask and a magnetic stir bar, and then cool slowly to room temperature in a desiccator over anhydrous calcium sulfate.

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2. 4-Aminothiol (2.00 g, 15.97 mmol) is dissolved in CH3CN (10 mL) and cool to 0  C in an ice-salt bath. 3. Add t-BuONO (2.84 mL, 23.90 mmol) and stir the mixture for 10 min at 0  C firstly, and then trimethylsilyl azide (2.53 mL, 19.17 mmol) is added dropwise for about 10 min via 5 mL syringe; the resulting brown solution is stirred at room temperature for 1 h. 4. One hour later the solvent is removed at reduced pressure (see Note 6) and the residue is purified by silica gel column chromatography using 25% EA in PE to afford the 4-azidobenzenethiol as a white solid (2.2 g, 91% yield) [24]. ESI-()-MS (M-H) is calculated for C6H5N3S: 150.02; found: 150.01. 3.2

Expression

1. Confirm the identity of all plasmids by sequencing. 2. Thaw electrocompetent BL21(DE3) cells on ice. Add 0.5–1 μL of pET22b containing the gene of interest, pBK-CysM-NtSat4 and pUltra-PhSeRS/tRNAtyr with a concentration between 60 and 100 ng/μL. Transfer the cells to a 0.1 cm electroporation cuvette and electroporate at 1800 V. Add 1 mL of 2YT media immediately. Incubate cells in a 37  C shaker for approximately 1 h. Plate 25–100 μL cells on LB plates containing 100 μg/mL ampicillin, 50 μg/mL spectinomycin, and 50 μg/ mL kanamycin (see Note 7). 3. After 8–10 h, pick two or three different single colonies into small-size volume cultures of 2YT media with 50 μg/mL ampicillin, 25 μg/mL spectinomycin, and 25 μg/mL kanamycin. Grow 10–15 hours at 37  C shaker. In two or three 1.5 mL EP tubes mix 500 μL grown 2YT media and 500 μL sterile 50% (v/v) glycerol in water. Store the cell stocks at 80  C, followed by freezing of cells in liquid nitrogen. 4. Dilute the cell culture in 1:50 to inoculate a large volume of fresh 2YT medium as you want with 100 μg/mL ampicillin, 50 μg/mL spectinomycin, and 50 μg/mL kanamycin. 5. Add the required thiol precursor to fresh 2YT expression medium with a final concentration of 1 mM when OD 0.6–0.8 reached. Meanwhile, add 0.5 mM IPTG to induce the expression of your interest proteins. Grow BL21(DE3) cells for an additional several hours at specific expression time and temperature based on your protein of interest. Harvest cells by centrifugation at 5000 g for 15 min. Decant supernatant (see Notes 8 and 9).

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Purification

1. Resuspend the cells in lysis buffer and incubate at 4  C for 2 h on shaker. For cytoplasmic protein expression and purification, the cell suspension is lysed by sonication (30% energy input, 10 min with 5-s pause after every 5-s sonication). For periplasmic protein expression and purification, the cell suspension is lysed by sonication (30% energy input, 1 min with 5-s pause after every 5-s sonication). 2. Centrifugate the lysate at 18,000 g for 30 min and incubate with Ni-NTA agarose at 4  C for 1 h. 3. Load the mixture onto a gravity column, wash 5–6 times with 10 mL of wash buffer. 4. Elute the protein of interest with 0.2–0.5 mL of elution buffer. 5. The purified proteins are exchanged to PBS buffer using Amicon Ultra column and store as glycerol stocks (10% glycerol in PBS) at 80  C. 6. The identities of purified proteins can be confirmed by SDS-PAGE and high-resolution electrospray-ionization mass spectrum.

3.4

Reaction

3.4.1 Conjugation Reaction of Azido Group Containing Proteins and DBCO-mPEG20K

1. The strain-promoted azide-alkyne cycloaddition (SPAAC) is performed in PBS. 2. The target protein with azido group is concentrated to 50 μM using appropriate cutoff centrifugal fiter device. 3. Dilute DBCO-mPEG20K in PBS to a concentration around 10–20 mM. 4. Mix the target protein with DBCO-mPEG20K at the molar ratio of 1:10–100. PBS as buffer is added to the final appropriate volume you want. Perform a quick vortex to ensure mixing. 5. The mixture is incubated at 37  C for 5–20 h. The reaction progress is monitored by SDS-PAGE with Coomassie staining (see Note 10). 6. Upon completion, purification can be performed by sizeexclusion chromatography or appropriate cutoff centrifugal filter device. 7. Determine the concentration of the conjugate, and store at 4  C for short-term use or as glycerol stocks (10% glycerol in PBS) at 80  C for long-term preservation.

3.4.2 Conjugation Reaction of Azido Group Containing Proteins and DBCO-CY5

1. The strain-promoted azide-alkyne cycloaddition (SPAAC) is performed in PBS. 2. Concentrate the target protein with an azido group to 50 μM using appropriate cutoff centrifugal fiter device. 3. Dilute DBCO-CY5 in PBS to a concentration around 10–30 mM.

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4. Mix the target protein with DBCO-CY5 at the molar ratio of 1: 3–10. Add PBS to the final appropriate volume you want. Perform a quick vortex to ensure mixing. 5. Incubate the mixture at 37  C for 5–20 h. Monitor the reaction progress by high-resolution electrospray-ionization mass spectrum (see Note 10). 6. Upon completion, purification can be performed by sizeexclusion chromatography or an appropriate cutoff centrifugal filter device. 7. Determine the concentration of the conjugate, and store at 4  C for short-term use or as glycerol stocks (10% glycerol in PBS) at 80  C for long-term preservation. 3.4.3 Conjugation Reaction of Ketone Group Containing Poteins and Hydroxyamine-mPEG20K

1. Perform oxime ligation in conjugation buffer. 2. Concentrate the target protein with a ketone group to 50 μM using an appropriate cutoff centrifugal fiter device. 3. Dilute hydroxyamine-mPEG20K in conjugation buffer to a concentration around 10–30 mM. 4. Mix the target protein with hydroxyamine-mPEG20K and aniline at the molar ratio of 1:30–50:0.1. Add the conjugation buffer to the final appropriate volume you want. Perform a quick vortex to ensure mixing. 5. Incubate the mixture at 37  C for 3–4 d. Monitor the reaction progress by SDS-PAGE with Coomassie staining (see Note 10). 6. Upon completion, purification can be performed by sizeexclusion chromatography or an appropriate cutoff centrifugal filter device. 7. Determine the concentration of the conjugate, and store at 4  C for short-term use or as glycerol stocks (10% glycerol in PBS) at 80  C for long-term preservation.

3.5

Selection

1. Generate a library of PhSeRS mutants by saturation mutagenesis at residues Leu65, Phe108, Gln109, and Asp158, where the codons of these positions are randomized to NNK (N ¼ A/T/ G/C, K ¼ T/G). 2. To transform the PhSeRS library into DH10B electrocompetent cells harboring a positive selection plasmid (pRep), add 100 ng library plasmid and mix the cells by tapping on the tube 5–6 times. Transfer the cells to a 0.2 cm electroporation cuvette and electroporate at 2500 V. Add 1 mL 2YT medium immediately and recover the cells at 37  C for 1 h. Plate the cells on 15 cm LB plates containing 10 μg/mL tetracycline and 50 μg/mL kanamycin. Grow the cells overnight and then scrape into 5 mL of 2YT. Dilute the cells with 5 mL of 50%

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glycerol, and store at 80  C in 1 mL aliquots for use in the first round of selections. 3. For the positive round of selection, dilute the glycerol preps to an appropriate spreading volume and plate on 15 cm GMML plates with 10 μg/mL tetracycline, 50 μg/mL kanamycin, 68 μg/mL chloramphenicol, and 1 mM thiol precursor. Incubate at 37  C until formation of lawns. Scrape the cells into 5 mL of 2YT, and collect the plasmid DNA using a kit. Isolate the PhSeRS library plasmids by 1% agarose gel purification with extraction of the plasmid DNA using a kit. 4. For the negetive round of selection, transform 100 ng purified DNA into DH10B electrocompetent cells harboring a negitive selection plamid (pNeg) in a 0.2 cm electroporation cuvette and electroporate at 2500 V. After transformations, recover the cells in 1 mL of 2YT at 37  C for 1 h. Plate the cells on 15 cm LB plates with 100 μg/mL ampicillin, 0.2% L-arabinose, and 50 μg/mL kanamycin. After overnight incubation, scrape lawns and isolate library plasmids same with positive selection procedures. 5. Several positive and negetive rounds are repeated (Fig. 4). Pick colonies from the last positive round and incubate in 96-well

Fig. 4 Selection process for evolving efficient aaRS variants recognizing the desired ncAAs biosynthesized from thiol precursors

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plates containing 400 μL GMML per well. Spot 2.5 μL from each well onto two sets of GMML plates with 10 μg/mL tetracycline, 50 μg/mL kanamycin, and 68 μg/mL chloramphenicol. In one set of the plates add 1 mM thio precursor and the synthetases in the cells which only grow on plates with thiol precursors are further evaluated.

4

Notes 1. pET22b is an isopropyl-β-D-1-thiogalactopyranosid-dependent expression plasmid that relies on the T7 promoter. pET22b is ampicillin-resistant. Your gene of interest could be cloned into the expression vector with an amber stop codon at the desired site. Inclusion of a 6
histidines tag at the C-terminus is needed for purification by Ni-NTA. 2. The CysM gene from the BL21 and NtSat4 gene [25] from Nicotiana tabacum is synthesized and codon optimized. They are expressed under the constitutive IPP and Trc1 promotors, respectively, in a pBK vector containing low copy number of p15a replicative origin and a kanamycin resistance marker to afford plasmid pBK-CysM-NtSat4. Flag peptide DYKDDDDK or 6
histidines tag was added at the C-terminus. DNA sequence of Ntsat4: ATGGCTGCGGCGACCCCGCCGAC CAACCCGCTGAGCCGTGACCCGAACAAGCCGCAGAT CGATAACCACGTGTACAACTACGTTAAGTTTTGCCGT CCGAGCTTTCCGGAACTGGTTAGCTGCGCGCCGATC CCGGAGAAGAACAGCAAAATTGGTCGTAACGAGGAA GAGGACGATCTGTGGCTGAAGATGAAAGACGAAGCG CGTAGCGACATCGATCAAGAGCCGATTCTGAGCACC TACTATATCACCAGCATTCTGGCGCACGATAGCATGG AACGTGCGCTGGCGAACCACCTGAGCATGAAGCTGA GCAACAGCAGCCTGCCGAGCAGCACCCTGTACGACC TGTTTCTGGGCGTGCTGACCGAAGATTGCAGCCAGG ATATCATTAAGGCGGTGATCGCGGACCTGCGTGCGG TTAAAGAGCGTGATCCGGCGTGCATTAGCTATGTTCA CTGCTTCCTGAACTTTAAGGGTTTCCTGGCGTGCCA AGCGCACCGTATCGCGCACAAACTGTGGAGCAACGG CCGTCAGATCCTGGCGCTGCTGATTCAAAACCGTGT TAGCGAAGTGTTCGCGGTTGACATTCACCCGGGTGC GAAGATCGGTAAAGGCATTCTGCTGGATCATGCGAC CGGTGTGGTTGTGGGTGAAACCGCGGTGATCGGCAA CAACGTTAGCATTCTGCACAACGTGACCCTGGGTGG CACCGGCAAGATCAGCGGCGACCGTCACCCGAAAAT CGGTGATGGCGTTCTGATTGGTGCGGGCACCTGCGT GCTGGGTAACGTTATCATTGAGGACGGCGCGAAAAT CGGTGCGGGCAGCGTTGTGCTGAAGAAAGTGCCGG CGCGTACCACCGCGGTTGGTAACCCGGCGCGTCTGC

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TGGGTGGCAAGGAAAACCCGAAGAAACTGGATAAAAT CCCGAGCCTGACCATGGACCACACCTACGAGTGGAG CGATTATGTTATTTAA. 3. pUltra-PhSeRS/tRNATyr is a spectinomycin-resistant plasmid containing M. jannaschii aaRS as well as the M. jannaschii tRNACUA evolved for the incorporation of diversified noncanonical amino acids with aromatic thiols. If you attempt to evolve a better aaRS mutant for the desired ncAAs, you could screen a synthetase library by simply feeding cells with the corresponding aromatic thiol precursor following the works from us [22] and others [26]. 4. These buffers are suitable for purifying the proteins we are interested in, but may not be compatible with other proteins of interest. Specific purification buffers should be adjusted as you needed. 5. Usually, five times the mass of silica was added in the bottom flask, and a mixture of crude product and silica was resuspend in EA. 6. Caution! Do not perform extraction. Adding the mixture to aqueous media would lead to formation of highly toxic N3H gases. Make sure to add silica gel to the mixture directly. 7. Please note that some cells have innate resistance to antibiotics. To avoid false positives during selection, please make sure that plasmids bear different replication origins to avoid plasmid loss. 8. The aromatic thiol concentration has some effect on cell growth owing to the toxicity of aromatic thiols. We suggest supplemention with 1 mM PhSNa, which has minimal influence on the growth of the E. coli BL21 cells. 9. Aromatic thiols can be smelly. We recommend to perform the experiments in a well-ventilated hood. 10. Reactions of DBCO with azide and ketone with hydroxyamine are bimolecular. Thus, the time required for full conversion depends on the concentrations of the reagents. References 1. Macek B, Forchhammer K, Hardouin J, Weber-Ban E, Grangeasse C, Mijakovic I (2019) Protein post-translational modifications in bacteria. Nat Rev Microbiol 17(11): 651–664. https://doi.org/10.1038/s41579019-0243-0 2. Dumas A, Lercher L, Spicer CD, Davis BG (2015) Designing logical codon reassignment – expanding the chemistry in biology. Chem Sci 6(1):50–69. https://doi.org/10.1039/ c4sc01534g

3. Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292(5516):498–500. https://doi.org/10.1126/science.1060077 4. Wang J, Wang X, Fan X, Chen PR (2021) Unleashing the power of bond cleavage chemistry in living systems. ACS Cent Sci 7(6): 9 2 9 – 9 4 3 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 1 / acscentsci.1c00124 5. Wang X, Liu D, Shen L, Li F, Li Y, Yang L, Xu T, Tao H, Yao D, Wu L, Hirata K, Bohn

Protein Expression with Biosynthesized Noncanonical Amino Acids LM, Makriyannis A, Liu X, Hua T, Liu Z-J, Wang J (2021) A genetically encoded F-19 NMR probe reveals the allosteric modulation mechanism of cannabinoid receptor 1. J Am Chem Soc 143(40):16320–16325. https:// doi.org/10.1021/jacs.1c06847 6. Park SH, Ko W, Park SH, Lee HS, Shin I (2019) Evaluation of the interaction between Bax and Hsp70 in cells by using a FRET system consisting of a fluorescent amino acid and YFP as a FRET pair. Chembiochem 21(1–2):59–63. https://doi.org/10.1002/cbic.201900293 7. Zhang B, Sun J, Wang Y, Ji D, Yuan Y, Li S, Sun Y, Hou Y, Li P, Zhao L, Yu F, Ma W, Cheng B, Wu L, Hu J, Wang M, Song W, Li X, Li H, Fei Y, Chen H, Zhang L, Tsokos GC, Zhou D, Zhang X (2021) Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells. Nat Biomed Eng 5(11): 1288–1305. https://doi.org/10.1038/ s41551-021-00797-8 8. Ptacin JL, Caffaro CE, Ma L, San Jose Gall KM, Aerni HR, Acuff NV, Herman RW, Pavlova Y, Pena MJ, Chen DB, Koriazova LK, Shawver LK, Joseph IB, Milla ME (2021) An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism. Nat Commun 12(1). https://doi.org/10.1038/ s41467-021-24987-9 9. Zhang J, Ji D, Shen W, Xiao Q, Gu Y, O’Shaughnessy J, Hu X (2022) Phase I trial of a novel anti-HER2 antibody–drug conjugate, ARX788, for the treatment of HER2positive metastatic breast cancer. Clin Cancer Res:OF1–OF10. https://doi.org/10. 1158/1078-0432.Ccr-22-0456 10. Ling X, Chang L, Chen H, Gao X, Yin J, Zuo Y, Huang Y, Zhang B, Hu J, Liu T (2021) Improving the efficiency of CRISPRCas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates. Mol Cell 81(22):4747–4756.e4747. https://doi.org/ 10.1016/j.molcel.2021.09.021 11. Ling X, Xie B, Gao X, Chang L, Zheng W, Chen H, Huang Y, Tan L, Li M, Liu T (2020) Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates. Sci Adv 6(15). https:// doi.org/10.1126/sciadv.aaz0051 12. Tang H, Dai Z, Qin X, Cai W, Hu L, Huang Y, Cao W, Yang F, Wang C, Liu T (2018) Proteomic identification of protein tyrosine phosphatase and substrate interactions in living mammalian cells by genetic encoding of irreversible enzyme inhibitors. J Am Chem Soc 140(41):13253–13259. https://doi.org/10. 1021/jacs.8b06922

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blocks with noncanonical amino acids biosynthesized from aromatic thiols. Angew Chem Int Ed 60(18):10040–10048. https:// doi.org/10.1002/anie.202014540 23. Liu Y, Kim J, Seo H, Park S, Chae J (2015) Copper(II)-catalyzed single-step synthesis of aryl thiols from aryl halides and 1,2-ethanedithiol. Adv Synth Catal 357(10): 2205–2212. https://doi.org/10.1002/adsc. 201400941 24. Brai A, Martelli F, Riva V, Garbelli A, Fazi R, Zamperini C, Pollutri A, Falsitta L, Ronzini S, Maccari L, Maga G, Giannecchini S, Botta M (2019) DDX3X helicase inhibitors as a new

strategy to fight the West Nile virus infection. J Med Chem 62(5):2333–2347. https://doi. org/10.1021/acs.jmedchem.8b01403 25. Wirtz M, Hell R (2003) Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase. Amino Acids 24(1): 195–203. https://doi.org/10.1007/s00726002-0313-9 26. Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79(1):413–444. https://doi.org/10. 1146/annurev.biochem.052308.105824

Chapter 7 Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli Han-Kai Jiang and Jeffery M. Tharp Abstract Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/ aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNAPyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons. Key words Genetic code expansion, Non-canonical amino acids, Pyrrolysyl-tRNA synthetase, Synthetic biology, Bioorthogonal chemistry

1

Introduction To date, more than 200 distinct noncanonical amino acids (ncAAs) have been installed into proteins in living cells by genetic code expansion [1–3]. This technique uses heterologously expressed, orthogonal aminoacyl-tRNA synthetase (aaRS) and tRNA pairs to site-specifically install ncAAs into proteins during ribosomal protein synthesis. The orthogonal aaRS recognizes a desired ncAA as a substrate and specifically aminoacylates the orthogonal tRNA with the ncAA. The resulting aminoacyl-tRNA then introduces the ncAA into proteins, in response to a repurposed codon, typically the amber stop codon UAG [4]. The ability to install ncAAs into proteins in this way has many useful applications including introducing reactive ncAAs into proteins for site-specific covalent modification [5, 6], producing designer enzymes with new functions [7, 8], generating genetically encoded cyclic peptides [9, 10],

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_7, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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synthesizing sequence-defined polymers [11, 12], and producing proteins with defined posttranslational modifications [13]. Using multiple, mutually orthogonal aaRS and tRNA pairs, multiple distinct ncAAs can be installed into proteins within the same cell. Typically, this is achieved using one orthogonal tRNA that recognizes the amber stop codon (UAG) and a second orthogonal tRNA recognizing the ochre stop codon (UAA) [5, 14, 15]. This leaves the remaining opal stop codon (UGA) to terminate translation of the target protein. Introducing more than two unique ncAAs into proteins is challenging owing of the lack of “blank” codons for encoding the ncAAs. While simultaneous reassignment of all three stop codons has been used to produce proteins containing three distinct ncAAs in bacteria and eukaryotes [6, 16], this strategy requires co-expression of TEV protease, which assists in terminating translation. Alternatively, four-base codons can be used, together with stop codons, to encode three or four ncAAs [17, 18]. Yet, decoding of four-base codons is inefficient, and tRNAs with four-base anticodons are often poorly recognized by aaRSs [19–21]. A third strategy for generating blank codons to encode ncAAs is to reassign a “sense” codon that already encodes a canonical amino acid. However, with this strategy, the orthogonal tRNA must compete with endogenous tRNAs for suppressing the sense codon. This often results in heterogeneous protein products containing a mixture of the canonical and noncanonical amino acids [22, 23]. Recently, we engineered a novel chimeric tRNA from the E. coli initiator tRNA and Methanocaldococcus jannaschii tyrosine tRNA [24, 25]. We demonstrated that this chimeric tRNA (itRNATy2) is efficiently aminoacylated with ncAAs by the M. jannaschii tyrosyltRNA synthetase (MjTyrRS). Once produced, the aminoacyl-itRNATy2 can initiate the synthesis of new polypeptides in response to the amber stop codon (Fig. 1a). We subsequently showed that itRNATy2 can be modified to recognize the UAU tyrosine codon and that the modified tRNA (itRNATy2AUA) can install ncAAs at the protein N-terminus in response to a UAU initiating codon [26]. By combining UAU initiation with stop codon suppression, itRNATy2AUA can be used to efficiently install three distinct ncAAs into proteins in E. coli (Fig. 1b). Here we describe a general protocol for introducing three distinct ncAAs into proteins in response to the UAU, UAG, and UAA codons. This protocol relies on three mutually orthogonal aaRS and tRNA pairs, including the MjTyrRS and itRNATy2AUA pair and the pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl pairs from Methanosarcina mazei (Mm) and Ca. Methanomethylophilus alvus (Ma). The following protocol describes the installation of three reactive ncAAs (Fig. 2) into a model, His-tagged, green fluorescent protein (sfGFP); however, the protocol can be adapted for any combination of three ncAAs that are substrates of MjTyrRS, MmPylRS, and MaPylRS, as well as for any protein of interest.

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Fig. 1 Genetic encoding of noncanonical amino acids (ncAAs) using an engineered initiator tRNA. (a) The engineered initiator tRNA (itRNATy2) can be used to install ncAAs at the N-terminus of proteins in response to a UAG initiating codon. (b) A modified initiator tRNA (itRNATy2AUA) can be used to install ncAAs at the N-terminus of proteins in response to a UAU initiating codon. The tRNA is aminoacylated by the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS). UAU initiation can be combined with UAG and UAA suppression to install three distinct ncAAs into proteins in response to UAU, UAG, and UAA codons. The UAG and UAA codons are suppressed by the pyrrolysyl-tRNA synthetase/tRNAPyl pairs from Ca. Methanomethylophilus alvus (Ma) and Methanosarcina mazei (Mm), respectively

2 Materials 2.1 Cotransformation

1. Lysogeny broth (LB): Add 4 g tryptone, 2 g yeast extract, and 4 g NaCl to 400 mL double-distilled water. Autoclaved before use. Store at room temperature. 2. LB agar: Add 6 g agar, 4 g tryptone, 2 g yeast extract, and 4 g NaCl to 400 mL double-distilled water. Autoclaved before use. Store at room temperature. 3. Sterile, round-bottom culture tubes.

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Fig. 2 The structures of the ncAAs used in this protocol. Para-acetyl-Lphenylalanine (pAcF) is a substrate of AzFRS.2.t1, a MjTyrRS variant [27]. Meta-azido-L-phenylalanine (mAzF) is a substrate of a MaPylRS variant bearing an N166S mutation [26]. Nε-Propargyl-L-lysine (PrK) is a substrate of wild-type MmPylRS

4. Sterile Petri dishes (100 mm × 15 mm). 5. Sterile cell spreaders. 6. Water bath set to 42 °C. Clean the water bath when mold is found. 7. Stationary incubator set to 37 °C. 8. Shaking incubator set to 37 °C. 9. Ice bucket filled with ice. 10. Amp stock: Dissolve 1 g ampicillin powder in 10 mL doubledistilled water to 100 mg/mL. Filter, split into aliquots, and store at -20 °C. 11. Sm stock: Dissolve 1 g spectinomycin powder in 10 mL double-distilled water to 100 mg/mL (see Note 1). Filter, split into aliquots, and store at -20 °C. 12. Cm stock: Dissolve 0.34 g chloramphenicol powder in 10 mL ethanol to 34 mg/mL. Filter, split into aliquots, and store at 20 °C. 13. E. coli DH10BΔmetZWV [24] chemical competent cells (100 μL per aliquot) (see Note 2). 14. Three-plasmid expression system: pULTRA-MmPylRSMmtRNAPylUUA, pSTART-AzFRS.2.t1-MaPylRS(N166S)MatRNA(6)PylCUA, and pBAD-sfGFPOpt[1TAT-135TAG151TAA]-itRNATy2AUA-MatRNA(6)PylCUA (Fig. 3). Store at -20 °C (see Note 3). 2.2 Protein Expression

1. 2xYT medium: Dissolve 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L double-distilled water. Autoclaved before use. Store at room temperature. 2. Sterile, round-bottom culture tubes. 3. Autoclaved shaker flasks (see Note 4). 4. Shaking incubator set to 37 °C (see Note 5).

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Fig. 3 (a) The three-plasmid system used to install three distinct ncAAs into proteins. tRNAPyl refers to a variant of the M. alvus tRNAPyl that is orthogonal to the MmPylRS [28]. tRNAPylUUA refers to a variant of the M. mazei tRNAPyl that was engineered to efficiently decode UAA codons [15]. (b) The DNA sequence of a gene encoding a hypothetical protein of interest. The Shine-Dalgarno sequence that precedes the TAT initiating codon is shown. The TAG and TAA codons act as elongating codons. TGA is used as a stop codon to terminate protein synthesis. A C-terminal His-tag should be added just prior to the TGA codon for protein purification

5. 20% arabinose: Dissolve 2 g L-arabinose in 5 mL doubledistilled water. Add water to a total volume of 10 mL. Filter, split into aliquots, and store at -20 °C. 6. 1 M IPTG: Dissolve 2.38 g isopropyl-β-D-1-thiogalactopyranoside in 10 mL double-distilled water. Filter, split into aliquots, and store at -20 °C. 7. 100 mM pAcF: Add 207 mg of para-acetyl-L-phenylalanine to 10 mL of 0.3 M NaOH with frequent vortex. Store at 4 °C. 8. 100 mM mAzF: Add 206 mg of meta-azido-L-phenylalanine to 10 mL of 0.3 M NaOH with frequent vortex. Store at 4 °C (see Note 6). 9. 100 mM PrK: Add 264 mg of Nε-propargyl-L-lysine to 10 mL of double-distilled water with frequent vortex. Store at 4 °C.

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2.3 Protein Purification

1. BugBuster 10× Protein Extraction Reagent (Millipore-Sigma). Store at room temperature. 2. Benzonase Nuclease (Millipore-Sigma). Store at -20 °C. 3. TALON Metal Affinity Resin (Takara Bio). Store at 4 °C. 4. Precision Plus Protein Dual Color Standards (Bio-Rad). Store at -20 °C. 5. Mini-PROTEAN TGX Precast Gels (Bio-Rad). Store at 4 °C. 6. Amicon Ultra Centrifugal Filters (10 kDa MWCO, MilliporeSigma). 7. Wash buffer: Dissolve 6.06 g Tris base, 29.22 g NaCl, and 0.68 g imidazole in 800 mL double-distilled water. Adjust the pH to 8 using HCl. Add water to a total volume of 1 L (50 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 8.0). Store at 4 °C. 8. Lysis buffer: Dilute one volume of BugBuster in nine volumes of wash buffer. Add Benzonase Nuclease to 25 U/mL. Make immediately before use. 9. Elution buffer: Dissolve 6.06 g Tris base, 5.84 g NaCl, and 17.02 g imidazole in 800 mL double-distilled water with stirring. Adjust the pH to 8 using HCl. Add water to a total volume of 1 L (50 mM Tris, 100 mM NaCl, 250 mM imidazole, pH 8.0). Store at 4 °C. 10. Reaction buffer: Dissolve 5.05 g Na2HPO4•7H2O, 0.85 g NaH2PO4•H2O, and 1.46 g NaCl in 800 mL double-distilled water with stirring. Adjust the pH to 7.4 using NaOH or HCl. Add water to a total volume of 1 L (25 mM sodium phosphate, 25 mM NaCl, pH 7.4). Store at room temperature (see Note 7). 11. Coomassie blue stain: Dissolve 2.5 g Coomassie Brilliant Blue R-250 in 250 mL methanol, 50 mL glacial acetic acid, and 200 mL distilled water. Filter the solution with Whatman No 1 filter. Store at room temperature. 12. Destaining solution: Mix 250 mL methanol, 50 mL glacial acetic acid, and 200 mL distilled water. Store at room temperature. 13. Empty gravity flow columns (Bio-Rad).

2.4

Protein Labeling

1. 10 mM 3-azido-7-hydroxycoumarin: Dissolve 20.3 mg 3-azido-7-hydroxycoumarin in 10 mL DMSO. Store at -20 ° C (see Note 8). 2. 10 mM Fluor 488-alkyne: Dissolve 2.9 mg Fluor 488-alkyne in 0.5 mL DMSO. Store at -20 °C. 3. 25 mM HiLyte Fluor 488-hydroxylamine: Dissolve 1 mg HiLyte Fluor 488-hydroxylamine in 82 μL DMSO. Store at -20 °C.

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4. 5 mM CuSO4: Prepare a 10× stock solution by dissolving 0.8 g CuSO4 in 100 mL of water. Dilute this solution tenfold in water to achieve a 5 mM working stock. Store at room temperature. 5. 10 mM BTTAA: Dissolve 43.05 mg BTTAA in 10 mL DMSO. Store at -20 °C. 6. 250 mM sodium ascorbate: Dissolve 0.495 g sodium ascorbate in 10 mL double-distilled water. Store at 4 °C (see Note 9). 7. Reaction buffer: Dissolve 5.05 g Na2HPO4•7H2O, 0.85 g NaH2PO4•H2O, and 1.46 g NaCl in 800 mL double-distilled water. Adjust the pH to 7.4 using NaOH or HCl. Add water to a total volume of 1 L (25 mM sodium phosphate, 25 mM NaCl, pH 7.4). Store at room temperature. 8. Low pH reaction buffer: Dissolve 13.6 g KH2PO4 and 14.6 g NaCl in 800 mL double-distilled water. Adjust the pH to 4.5 using HCl. Add water to a total volume of 1 L (100 mM potassium phosphate, 250 mM NaCl, pH 4.5). Store at room temperature. 9. Amicon Ultra-0.5 Centrifugal Filter Units (3 kDa MWCO, Millipore-Sigma).

3

Methods

3.1 Cotransformation

1. Thaw an aliquot of E. coli DH10BΔmetZWV chemical competent cells on ice for 10 min. 2. Add 1 μL each of pULTRA-MmPylRS-MmtRNAPylUUA, pSTART-AzFRS.2.t1-MaPylRS(N166S)-MatRNA(6)PylCUA, and pBAD-sfGFPOpt[1TAT-135TAG-151TAA]-itRNATy2AUAMatRNA(6)PylCUA to the competent cells and mix by gently pipetting. Incubate the plasmid-cell mixture on ice for 20 min (see Note 10). 3. Incubate the plasmid-cell mixture in a water bath at 42 °C for 60 s. 4. Put the transformed mixture immediately back on ice and incubate for an additional 2 min. 5. Add 1 mL of LB medium to the transformed mixture. Incubate at 37 °C and 200 rpm for 1 h. 6. In parallel, prepare an LB agar plate containing Amp, Sm, and Cm. Completely melt the LB agar using a microwave and then cool the molten agar to 60 °C. Add 20 μL of Amp stock, 10 μL of Sm stock, and 20 μL of Cm stock to a sterile 50 mL centrifuge tube (see Note 11). Pour 20 mL of the molten LB agar into the tube and gently invert several times. Pour the mixture

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into a sterile 100 mm × 15 mm petri dish and avoid introducing bubbles into the molten agar. Put the lid on top of the plate until the agar has solidified. Dry the plate in a laminar flow hood for 15 min before use. 7. Centrifuge the transformed cell mixture for 2 min at 8500×g. 8. Discard 1 mL of supernatant and gently resuspend the cell pellet using the remaining 100 μL of media. 9. Transfer 100 μL of the cell suspension to the surface of the LB agar plate and spread the cells evenly across the surface of the plate until dry. 10. Incubate the plate upside down at 37 °C overnight (see Note 12). 3.2 Protein Expression

1. Pick a single colony from the plate containing the co-transformed cells and inoculate 10 mL of 2xYT media containing 10 μL of Amp stock, 5 μL of Sm stock, and 10 μL of Cm stock. 2. Incubate at 37 °C and 200 rpm overnight. 3. Dilute the overnight cell culture 1:50 in 400 mL of autoclaved 2xYT media supplemented with 400 μL of Amp stock, 200 μL of Sm stock, and 400 μL of Cm stock. The media should be prepared in a 2-L shaker flask. 4. Incubate at 37 °C and 200 rpm until OD600 reaches 0.3 to 0.5. 5. Add 400 μL of 1 M IPTG, 4 mL of 20% arabinose, and 8 mL each of the 100 mM PrK, pAcF, and mAzF stocks to the culture flask (see Note 13). 6. Incubate at 37 °C and 200 rpm for 18 to 20 h. 7. Centrifuge the cells at 4 °C and 8500×g and collect the pellet in a sterile centrifuge tube. Purify the protein right away or freeze the pellet using liquid nitrogen and store at -80 °C.

3.3 Protein Purification

1. Resuspend the pellet in 20 mL lysis buffer. Lyse the cells by incubating the cell suspension at room temperature for 30 min with gentle shaking. 2. Centrifuge the cell lysate at 4 °C and 10,000×g for 30 min. Collect the supernatant and place on ice. 3. Pack 1 mL of TALON Metal Affinity Resin into a gravity flow column. Pass 5 mL of wash buffer through the packed column and discard the flow-through. 4. Add 20 mL of cleared supernatant to the packed column. Collect the flow-through for SDS-PAGE analysis. 5. Add 10 mL of wash buffer to the packed column to wash away nonspecifically bound proteins. Collect the flow-through for SDS-PAGE analysis.

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6. Add 5 mL of elution buffer to the packed column to elute the target protein. Collect the flow-through for SDS-PAGE analysis. 7. Add 5 mL of the eluate to an Amicon Ultra 10-kDa device. Fill the device to 15 mL with reaction buffer. Concentrate the sample down to 500 μL by centrifugation. Repeat this process at least three times (see Note 14). 8. Transfer the concentrated sample from the device to a sterile 1.5 mL tube. 9. Analyze the concentrated protein by SDS-PAGE and stain the gel with Coomassie blue for 1 h with gentle shaking. 10. Submerge the gel in destaining solution. Incubate at room temperature with gentle shaking. Destaining may need to be repeated several times until the background of the gel can be clearly seen. 11. Determine protein concentration by measuring the absorbance at 280 nm. 12. The successful incorporation of all three ncAAs into the protein can be confirmed by high-resolution mass spectrometry (Fig. 4a, b). The site specificity of each ncAA should be further confirmed using tandem mass spectrometry. 3.4 Protein Labeling by Copper-Catalyzed Azide-Alkyne Cycloaddition

1. Preform the copper-BTTAA complex by adding 2 μL of CuSO4 and 2 μL of BTTAA to 92 μL of reaction buffer. Mix by pipetting the solution up and down several times. 2. To the above mixture add 100 μL of purified protein (20 μM in reaction buffer). Mix by pipetting the solution up and down several times. 3. Depending on the desired labeling reaction, add either 2 μL of 3-azido-7-hydroxycoumarin or 2 μL of Fluor 488-alkyne to the above mixture. Mix by pipetting up and down several times. 4. Add 2 μL of sodium ascorbate to the above solution and mix by pipetting. 5. Incubate the reaction at room temperature, in the dark, for 1 to 2 h (see Note 15). 6. To remove excess dye and reagents, dilute the reaction to 500 μL using reaction buffer, and then transfer the diluted solution to an Amicon Ultra-0.5 Centrifugal Filter Unit. Centrifuge to concentrate the solution to 100 μL. Repeat this process at least three times. 7. Resolve the labeled protein using SDS-PAGE and take in-gel fluorescence images using a gel imager (e.g., Bio-Rad ChemiDoc gel imaging system; Fig. 4).

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Fig. 4 Analysis of proteins containing three ncAAs. (a) A cartoon representation of sfGFP harboring the ncAAs pAcF, mAzF and PrK. (b) Representative mass spectrometry of a protein containing three ncAAs. The intact mass can confirm that all three ncAAs are incorporated. The site specificity of ncAAs should be further confirmed by tandem mass spectrometry of the fragmented protein. Cal = calculated mass, Obs = observed mass. (c) Fluorescence labeling of sfGFP containing pAcF, mAzF, and PrK. Adapted with permission from Tharp, J. M., Vargas-Rodriguez, O., Schepartz, A., So¨ll, D. Genetic encoding of three distinct noncanonical amino acids using reprogrammed initiator and nonsense codons. (ACS Chem. Biol. 16(4), 766–774 (2021). Copyright 2022 American Chemical Society) 3.5 Protein Labeling by Oxime Ligation

1. Dilute 25 μL of purified protein (40 μM in reaction buffer) with 75 μL of low-pH reaction buffer. 2. Add 2 μL of HiLyte Fluor 488-hydroxylamine and incubate at 25 °C for 18 to 24 h (see Note 16). 3. Remove unreacted dye by filtering the protein using an Amicon Ultra-0.5 Centrifugal Filter Unit as described in Subheading 3.4. 4. Analyze the Protein by SDS-PAGE as Described in Subheading 3.4.

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Notes 1. At first, spectinomycin may not fully dissolve after thawing stock solutions. Once thawed, vigorously vortex to ensure that the solid is thoroughly dissolved. 2. The E. coli strain DH10BΔmetZWV was developed for improved translation initiation with ncAAs. Please contact Dr. Jeffery M. Tharp for information on how to obtain this strain. Nevertheless, the system would also work in other strains of E. coli, albeit the efficiency may be lower. 3. The protocol can be adapted for use with other aaRS mutants that recognize different ncAAs. This protocol utilizes the MjTyrRS mutant AzFRS.2.t1 which recognizes the ncAA pAcF [27], an MaPylRS variant harboring an N166S mutation which recognizes the ncAA mAzF [26], and wild-type MmPylRS which recognizes the ncAA PrK. The DNA sequence for these aaRSs and tRNAs is given below: MjTyrRS variant AzFRS.2.t1: ATGGACGAATTTGAAATGATAAAGAGAAACACATCT GAAATTATCAGCGAGGAAGAGTTAAGAGAGGTTT TAAAAAAGGATGAAAAATCTGCTCTGATAGGTTT TGAACCAAGTGGTAAAATACATTTAGGGCATTAT CTCCAAATAAAAAAGATGATTGATTTACAAAATGC TGGATTTGATATAATTATATTGTTGGCTGATTTAC ACGCCTATTTAAACCAGAAAGGAGAGTTGGATGA GATTAGAAAAATAGGAGATTATAACAAAAAAGTTT TTGAAGCAATGGGGTTAAAGGCAAAATATGTTTA TGGAAGTACTTATATGCTTGATAAGGATTATACA CTGAATGTCTATAGATTGGCTTTAAAAACTACCT TAAAAAGGGCAAGAAGGAGTATGGAACTTATAGC AAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTTGTCAT TATAGGGGCGTTGATGTTGCTGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAG CTTTTACCAAAAAAGGTTGTTTGTATTCACAACC CTGTCTTAACGGGTTTGGATGGAGAAGGAAAG ATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTG ATGACTCTCCAGAAGAGATTAGGGCTAAGATAAA GAAAGCATACTGCCCAGCTGGAGTTGTTGAAGG AAATCCAATAATGGAGATAGCTAAATACTTCCTTG AATATCCTGGTGGAGATTTGACAGTTAATAGCTA TGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAA TTGCATCCAATGCGCTTAAAAAATGCTGTAGCTG AAGAACTTATAAAGATTTTAGAGCCAATTAGAAA GAGATTATAA.

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MaPylRS(166S): ATGACAGTCAAATACACCGATGCACAGATTCAGCG TCTGCGTGAATATGGTAATGGCACCTATGAACAG AAAGTGTTTGAAGATCTGGCAAGCCGTGATGCA GCATTTAGCAAAGAAATGAGCGTTGCAAGCACC GACAATGAGAAAAAAATCAAAGGCATGATTGCAA ACCCGAGCCGTCATGGTCTGACCCAGCTGATGA ATGATATTGCAGATGCACTGGTTGCCGAAGGTT TTATTGAAGTTCGTACCCCGATTTTCATCAGCAA AGATGCCCTGGCACGTATGACCATTACCGAAGAT AAACCGCTGTTCAAACAGGTGTTTTGGATTGATG AAAAACGTGCACTGCGTCCGATGCTGGCACCGA ATCTGTATAGCGTTATGCGTGATCTGCGCGATC ATACCGATGGTCCGGTTAAAATCTTTGAAATGGG TAGCTGCTTTCGCAAAGAAAGCCATAGCGGTAT GCATCTGGAAGAATTTACCATGCTGTCACTGGT AGATATGGGTCCGCGTGGTGATGCAACCGAAGT TCTGAAAAACTATATTAGCGTTGTGATGAAAGCA GCAGGTCTGCCGGATTATGATCTGGTTCAAGAA GAAAGCGACGTCTACAAAGAAACCATTGATGTG GAAATTAACGGCCAAGAAGTTTGTAGCGCAGCA GTTGGTCCGCATTATCTGGATGCAGCACATGAT GTGCATGAACCGTGGTCAGGTGCAGGTTTTGGT CTGGAACGTCTGCTGACCATTCGTGAGAAATAT AGCACCGTTAAAAAAGGTGGTGCGAGCATTAGC TATCTGAATGGTGCCAAGATCAATTGA. MmPylRS: ATGGATAAAAAACCACTAAACACTCTGATATCTGCA ACCGGGCTCTGGATGTCCAGGACCGGAACAATTCATAAAATAAAACACCACGAAGTCTCTCGAAGCA AAATCTATATTGAAATGGCATGCGGAGACCACCT TGTTGTAAACAACTCCAGGAGCAGCAGGACTGC AAGAGCGCTCAGGCACCACAAATACAGGAAGAC CTGCAAACGCTGCAGGGTTTCGGATGAGGATCTCAATAAGTTCCTCACAAAGGCAAACGAAGACCAGACAAGCGTAAAAGTCAAGGTCGTTTCTGCCCCTACCAGAACGAAAAAGGCAATGCCAAAATCCGTT GCGAGAGCCCCGAAACCTCTTGAGAATACAGAA GCGGCACAGGCTCAACCTTCTGGATCTAAATTTT CACCTGCGATACCGGTTTCCACCCAAGAGTCAG TTTCTGTCCCGGCATCTGTTTCAACATCAATATC AAGCATTTCTACAGGAGCAACTGCATCCGCACT GGTAAAAGGGAATACGAACCCCATTACATCCATG TCTGCCCCTGTTCAGGCAAGTGCCCCCGCACTT ACGAAGAGCCAGACTGACAGGCTTGAAGTCCTG TTAAACCCAAAAGATGAGATTTCCCTGAATTCCG GCAAGCCTTTCAGGGAGCTTGAGTCCGAATTGC

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TCTCTCGCAGAAAAAAAGACCTGCAGCAGATCTA CGCGGAAGAAAGGGAGAATTATCTGGGGAAACT CGAGCGTGAAATTACCAGGTTCTTTGTGGACAG GGGTTTTCTGGAAATAAAATCCCCGATCCTGATC CCTCTTGAGTATATCGAAAGGATGGGCATTGATA ATGATACCGAACTTTCAAAACAGATCTTCAGGGT TGACAAGAACTTCTGCCTGAGACCCATGCTTGC TCCAAACCTTTACAACTACCTGCGCAAGCTTGAC AGGGCCCTGCCTGATCCAATAAAAATTTTTGAAA TAGGCCCATGCTACAGAAAAGAGTCCGACGGCA AAGAACACCTCGAAGAGTTTACCATGCTGAACTT CTGCCAGATGGGATCGGGATGCACACGGGAAAA TCTTGAAAGCATAATTACGGACTTCCTGAACCAC CTGGGAATTGATTTCAAGATCGTAGGCGATTCCT GCATGGTCTATGGGGATACCCTTGATGTAATGCA CGGAGACCTGGAACTTTCCTCTGCAGTAGTCGG ACCCATACCGCTTGACCGGGAATGGGGTATTGA TAAACCCTGGATAGGGGCAGGTTTCGGGCTCGA ACGCCTTCTAAAGGTTAAACACGACTTTAAAAAT ATCAAGAGAGCTGCAAGGTCCGAGTCTTACTATA ACGGGATTTCTACCAACCTGTAA. itRNATy2AUA: CGCGGGGTGGAGCAGCCTGGTAGCTCGTCGGGCT ATAAACCCGAAGATCGTCGGTTCAAATCCGGCC C CCGCGACCA. MatRNA(6)Pyl: GGGGGACGGTCCGGCGACCAGCGGGTCTCTA AAA CCTAGCATAGCGGGGTTCGACACCCCGGTCTCTC GCCA. MmtRNAPylUUA: GGAAACCTGATCATGTAGATCGAACGGACT TTA AA TCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCG CCA 4. To ensure adequate gas exchange, the volume of shaker flasks should be at least fivefold greater than the volume of the culture medium. To avoid contamination, the flask should be covered with foil, or an autoclavable cap at all times. 5. The expression temperature can be adjusted up or down depending on the needs of the target protein that is being expressed. 6. Azide functionality on ncAAs can be reduced to amines. To avoid this undesired reaction, prepare buffers without reducing agents. 7. This buffer is used for subsequent labeling of the protein by copper-catalyzed azide-alkyne cycloaddition. If the protein will

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not be immediately labeled, storage buffer and purification conditions should be adjusted to suit the needs of the target protein. 8. In general, all fluorescent dyes should be protected from light. We prepare solutions in 1.5 mL tubes and keep the tubes wrapped in aluminum foil. When not in use, store the dyes in the dark at -20 °C. 9. The sodium ascorbate solution should be prepared fresh on the day that the labeling reaction will be carried out. We have found that old solutions result in dramatically reduced labeling efficiency. 10. Plasmid concentrations for transformation of three plasmids typically range from 25 to 100 ng/μL. 11. For this three-plasmid system, the optimum final concentrations of antibiotics are Amp, 100 μg/mL; Sm, 50 μg/mL; and Cm, 34 μg/mL. 12. DH10BΔmetZWV have a modest growth reduction. With three antibiotics, it will generally take 20–24 h before reasonably sized colonies have grown on the plate. 13. In general, we use a final concentration of 2 mM of each ncAA. For more precious ncAAs, a lower concentration can be used. The concentration may need to be optimized for different ncAAs. Avoid adding too much NaOH to the culture which will inhibit cell growth. 14. The Amicon centrifugation device should be rinsed with reaction buffer before use. 15. The final concentrations of reaction components are as follows: 10 μM of the protein to be labeled, 50 μM CuSO4, 100 μM BTTAA, 100 μM fluorescent dye, and 2.5 mM sodium ascorbate. 16. The final concentrations of reaction components are as follows: 10 μM of the protein to be labeled and 500 μM of the hydroxylamine dye.

Acknowledgments The methods described in this protocol were developed in collaboration with the Center for Genetically Encoded Materials, an NSF Center for Chemical Innovation (CHE-2002182). The authors thank Professors Alanna Schepartz and Dieter So¨ll for valuable advice on preparing the manuscript. Han-Kai Jiang holds a graduate student fellowship from the Taiwan Academic Talents Overseas Advancement Program from the Ministry of Science and Technology (MOST 110-2917-I-007-006). Jeffery M. Tharp is supported

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pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids. Nat Chem 12(6): 535–544. https://doi.org/10.1038/s41557020-0472-x 18. Dunkelmann DL, Oehm SB, Beattie AT, Chin JW (2021) A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat Chem 13(11):1110–1117. https://doi.org/10.1038/s41557-02100764-5 19. Neumann H, Wang K, Davis L, Garcia-Alai M, Chin JW (2010) Encoding multiple unnatural amino acids via evolution of a quadrupletdecoding ribosome. Nature 464(7287): 4 4 1 – 4 4 4 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature08817 20. Wang N, Shang X, Cerny R, Niu W, Guo J (2016) Systematic evolution and study of UAGN decoding tRNAs in a genomically recoded bacteria. Sci Rep 6:21898. https:// doi.org/10.1038/srep21898 21. DeBenedictis EA, So¨ll D, Esvelt KM (2022) Measuring the tolerance of the genetic code to altered codon size. elife 11:e76941. https://doi.org/10.7554/eLife.76941 22. Krishnakumar R, Ling J (2014) Experimental challenges of sense codon reassignment: an innovative approach to genetic code expansion. FEBS Lett 588(3):383–388. https://doi.org/ 10.1016/j.febslet.2013.11.039 23. Schwark DG, Schmitt MA, Biddle W, Fisk JD (2020) The influence of competing tRNA abundance on translation: quantifying the

efficiency of sense codon reassignment at rarely used codons. Chembiochem 21(16): 2274–2286. https://doi.org/10.1002/cbic. 202000052 24. Tharp JM, Ad O, Amikura K, Ward FR, Garcia EM, Cate JHD, Schepartz A, So¨ll D (2020) Initiation of protein synthesis with non-canonical amino acids in vivo. Angew Chem Int Ed Engl 59(8):3122–3126. https://doi.org/10.1002/anie.201914671 25. Tharp JM, Krahn N, Varshney U, So¨ll D (2020) Hijacking translation initiation for synthetic biology. Chembiochem 21(10): 1387–1396. https://doi.org/10.1002/cbic. 202000017 26. Tharp JM, Vargas-Rodriguez O, Schepartz A, So¨ll D (2021) Genetic encoding of three distinct noncanonical amino acids using reprogrammed initiator and nonsense codons. ACS Chem Biol 16(4):766–774. https://doi.org/ 10.1021/acschembio.1c00120 27. Amiram M, Haimovich AD, Fan C, Wang Y-S, Aerni H-R, Ntai I, Moonan DW, Ma NJ, Rovner AJ, Hong SH, Kelleher NL, Goodman AL, Jewett MC, So¨ll D, Rinehart J, Isaacs FJ (2015) Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol 33(12):1272–1279. https://doi.org/10. 1038/nbt.3372 28. Willis JCW, Chin JW (2018) Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat Chem 10(8):831–837. https://doi.org/ 10.1038/s41557-018-0052-5

Chapter 8 Encoding Noncanonical Amino Acids into Phage Displayed Proteins Cristina Dı´az-Perlas, Montserrat Escobar-Rosales, Charles W. Morgan, and Benjamı´ Oller-Salvia Abstract Phage display facilitates the evolution of peptides and proteins for affinity selection against targets, but it is mostly limited to the chemical diversity provided by the naturally encoded amino acids. The combination of phage display with genetic code expansion allows the incorporation of noncanonical amino acids (ncAAs) into proteins expressed on the phage. In this method, we describe incorporation of one or two ncAAs in a single-chain fragment variable (scFv) antibody in response to amber or quadruplet codon. We take advantage of the pyrrolysyl–tRNA synthetase/tRNA pair to incorporate a lysine derivative and an orthogonal tyrosyl–tRNA synthetase/tRNA pair to incorporate a phenylalanine derivative. The encoding of novel chemical functionalities and building blocks in proteins displayed on phage provides the foundation for further phage display applications in fields such as imaging, protein targeting, and the production of new materials. Key words Bioorthogonal reactions, Cyclopropene, Phage display, Protein engineering, Site-specific bioconjugation

1

Introduction Phage display [1] is a powerful technique that facilitates the selection of peptides and proteins binding to a specific target [2– 5]. However, a drawback of this technique is the limitation to the 20 canonical amino acids, with few exceptions including the incorporation of selenocysteine [6] or methionine analogues [7]. Several adaptations of phage display have expanded the amino acid diversity by chemical or enzymatic modification of amino acid side chains before affinity selection [8, 9]. Furthermore, advances in genetic code expansion [10–12] have enabled the incorporation of noncanonical amino acids (ncAAs) into proteins displayed on phage [9, 13–19]. Typically, only one ncAA is introduced on peptides or proteins displayed on phage to create chemical handles to facilitate

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_8, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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the cyclization of peptides or to incorporate small molecules to facilitate and guide the affinity selection. However, we have recently shown that two ncAAs can be encoded on the same phagedisplayed protein, enabling phage modification with two mutually orthogonal reactions [20]. Most approaches use variants of Methanococcus jannaschii tyrosyl–tRNA synthetase (MjTyrRS)/tRNACUA pair, which is orthogonal to endogenous synthetase/tRNA pairs in Escherichia coli [13–19]. This system has been used for the cyclization of peptides [13, 14, 19, 21] and even to evolve antibody fragments with chemical warheads [15, 16]. To expand the diversity of ncAAs in phage display, we have recently reported the application of the pyrrolysyl–tRNA synthetase (PylRS)/tRNA pair. This pair has been extensively used for the incorporation of diverse aliphatic ncAAs into proteins [22], and its use in introducing diversity of gene 3 of M13 phage has been demonstrated in a continuous evolution approach [23–25]. Moreover, ncAAs can be incorporated in response to both amber and quadruplet codons with the use of an evolved orthogonal ribosome [12], facilitating the combination with other orthogonal pairs. In this protocol (Fig. 1), based on our recent work [20], we describe the use of PylRS/tRNAUACU to efficiently incorporate Nε[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-L-lysine (CypK) on a single-chain fragment variable (scFv) antibody displayed on the phage surface. Incorporation of CypK enables the rapid modification of phage via an inverse electron-demand Diels– Alder cycloaddition with tetrazine-bearing molecules. We also explain how to incorporate both CypK and p-propargyloxy-L-phenylalanine (PrpF) on the same scFv, which enables dual modification of the displayed scFv with distinct probes, through mutually bioorthogonal conjugations, in a one-pot procedure.

2

Materials All methods performed with commercial kits are conducted using recommended manufacturers’ protocols. All glassware and pipette tips should be sterilized before use.

2.1 Cloning of the Target ScFv into the Phagemid

1. Gene block encoding the target scFv with 5′ NcoI and 3′ SacI sites (see Note 1, Table 1). 2. O-scFv–g3 phagemid [20] (Table 1). 3. Thermal block. 4. Restriction enzymes (e.g., NcoI and SacI as described in step 1). 5. T4 ligase. 6. DNA purification kit. 7. DNA gel extraction kit.

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Fig. 1 Scheme of the expression process and analysis to encode noncanonical amino acids (ncAAs) into displayed proteins. Schematic representations of the plasmids are shown as well as the necessary steps to achieve: the cloning of the scFv into the phagemid, the phage expression with ncAAs and their conjugation to fluorophores. The characterization of the expressed scFv is performed using ELISA, western blot, and in-gel fluorescence

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Table 1 Plasmid sequences Element

Sequence

Phagemid Orthogonal Shine– Dalgarno sequence

CGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAAT GCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCG CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTT ACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGCTC GAGACAATTTTCATATCCCTCCGCAA

PelB

ATGAAATACCTATTGCCTACGGCGGCCGCTGGATTGTTATTACTCGCGGC CCAGCCGGCCATGGCG

scFv (4D5)

GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCGAGCGTGGGCG ATCGTGTTACTATCACCTGTCGTGCCTCTCAGGACGTTAACACCGCGGT AGCATGGTACCAGCAGAAACCGGGTAAGGCTCCAAAACTGCTGATTTAT TCCGCGTCCTTTCTGTACTCTGGCGTTCCGAGCCGTTTCTCTGGTTCT CGTTCCGGCACTGATTTCACTCTGACCATCTCTTCTCTGCAACCGGAA GACTTCGCGACCTACTATTGCCAGCAACATTACACCACCCCACCTACTT TCGGCCAGGGTACCAAAGTAGAAATCAAGCGTACGGTAGCTGGTGGCG GTGGTTCCGGTGGCGGTGGCAGCGGTGGCGGCGGTTCTGGTGGCGG CGGCTCCGAAGTTCAACTGGTTGAGTCCGGCGGTGGTCTGGTGCAGC CGGGCGGTAGCCTGCGCCTGTCTTGCGCTGCGTCCGGCTTCAACATTA AAGACACCTACATTCACTGGGTCCGTCAGGCTCCGGGCAAGGGTCTGG AATGGGTAGCGCGCATCTACCCGACCAACGGTTATACCCGCTACGCAGA TTCTGTTAAAGGTCGCTTTACCATTTCCGCAGACACCAGCAAAAACACC GCTTACCTGCAAATGAATTCTCTGCGCGCAGAAGATACTGCTGTATATTAC TGTAGCCGTTGGGGCGGTGATGGTTTCTACGCAATGGATTACTGGGGTC AGGGTACTCTGGTTACTGTGA GCTCT

scFv–g3 spacer, Myc, and HA tags

GGATCCGAACAAAAACTCATCTCAGAAGAGGATCTGGGTAGCGCACGTC GGGCAGGTTCA TATCCGTATGATGTTCCGGATTATGCAAGCGGT

pAux Relevant AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAAC sequence in ACATGCAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAG orthogonal TGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATA 16S ACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGG GGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTA GTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAG AGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGG AGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCA TGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGG GAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAA GAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGGAAAACGGAGGGTG CAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGT TAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATA CTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCG GTGAAATGCGTAGAGATCTGGAGGAATACCG (continued)

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Table 1 (continued) Element

Sequence

Pyrrolysyl tRNA synthetase and tRNA

TCGGGAGTTGTCAGCCTGTCCCGCTTATAAGATCATACGCCGTTATACGTTG TTTACGCTTTGAGGAATCCCATATGATGGATAAAAAACCGCTGGATGTGC TGATTAGCGCGACCGGCCTGTGGATGAGCCGTACCGGCACCCTGCATAA AATCAAACATCATGAAGTGAGCCGCAGCAAAATCTATATTGAAATGGCGTG CGGCGATCATCTGGTGGTGAACAACAGCCGTAGCTGCCGTACCGCGCGT GCGTTTCGTCATCATAAATACCGCAAAACCTGCAAACGTTGCCGTGTGAG CGATGAAGATATCAACAACTTTCTGACCCGTAGCACCGAAAGCAAAAACA GCGTGAAAGTGCGTGTGGTGAGCGCGCCGAAAGTGAAAAAAGCGATGCC GAAAAGCGTGAGCCGTGCGCCGAAACCGCTGGAAAATAGCGTGAGCGCG AAAGCGAGCACCAACACCAGCCGTAGCGTTCCGAGCCCGGCGAAAAGCA CCCCGAACAGCAGCGTTCCGGCGTCTGCGCCGGCACCGAGCCTGACCCG CAGCCAGCTGGATCGTGTGGAAGCGCTGCTGTCTCCGGAAGATAAAATTA GCCTGAACATGGCGAAACCGTTTCGTGAACTGGAACCGGAACTGGTGAC CCGTCGTAAAAACGATTTTCAGCGCCTGTATACCAACGATCGTGAAGATT ATCTGGGCAAACTGGAACGTGATATCACCAAATTTTTTGTGGATCGCGGC TTTCTGGAAATTAAAAGCCCGATTCTGATTCCGGCGGAATATGTGGAACGT ATGGGCATTAACAACGACACCGAACTGAGCAAACAAATTTTCCGCGTGGA TAAAAACCTGTGCCTGCGTCCGATGCTGGCCCCGACCCTGTATAACTATC TGCGTAAACTGGATCGTATTCTGCCGGGTCCGATCAAAATTTTTGAAGTG GGCCCGTGCTATCGCAAAGAAAGCGATGGCAAAGAACACCTGGAAGAATT CACCATGGTTAACTTTTGCCAAATGGGCAGCGGCTGCACCCGTGAAAACC TGGAAGCGCTGATCAAAGAATTCCTGGATTATCTGGAAATCGACTTCGAA ATTGTGGGCGATAGCTGCATGGTGTATGGCGATACCCTGGATATTATGCA TGGCGATCTGGAACTGAGCAGCGCGGTGGTGGGTCCGGTTAGCCTGGAT CGTGAATGGGGCATTGATAAACCGTGGATTGGCGCGGGTTTTGGCCTGG AACGTCTGCTGAAAGTGATGCATGGCTTCAAAAACATTAAACGTGCGAGC CGTAGCGAAAGCTACTATAACGGCATTAGCACGAACCTGTAACTGCAGTT TCAAACGCTAAATTGCCTGATGCGCTACGCTTATCAGGCCTACATGATCTC TGCAATATATTGAGTTTGCGTGCTTTTGTAGGCCGGATAAGGCGTTCACG CCGCATCCGGCAAGAAACAGCAAACAATCCAAAACGCCGCGTTCGGCGG TCGACACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATG CAGATCCGGAACATAATGGTGCAGGGCGCTTGTTTCGGCGTGGGTATGG TGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCTGCCGGCACCTGTC CTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTC ATGCCCCGCGCCCACCGGAAGGAGCTACCGGCAGCGGTGCGGACTGTTG TAACTCAGAATAAGAAATGAGGCCGCTCATGGCGTTCTGTTGCCCGTCTC ACTGGTGAAAAGAAAAACAACCCTGGCGCCGCTTCTTTGAGCGAACGATC AAAAATAAGTGGCGCCCCATCAAAAAAATATTCTCAACATAAAAAACTTTGT GTAATACTTGTAACGCTAGATCTGGAAACCTGATCATGTAGATCGAATTGG CTTACTATCCTGTTCAGCCGGGTTAGATTCCCGGGGTTTCCGCCAACTAG TATCCTTAGCGAAAGCTAAGGATTTTTTTTAAGCTTGGCACTGGCCGTCGT TTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCC TTGCAGCACATCCCCCTTTCGCCAGACGCTCTCCCTTATGCGACTCCTGC ATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCG CAAGGAATGGTGCATGCAAGGAGCCCGAGATGCGCCGCGTGCGGCTGCT GGAGATGGCGGACGCGATGGATATGTTCTGGCGGCCGC

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2.2 Phage Expression with ncAAs

1. 2xYT medium. 2. LB agar plates. 3. Antibiotics: ampicillin (Amp), kanamycin (Kan), spectinomycin (Spec), and tetracycline (Tet). 4. Shaking thermostatic incubator. 5. UV/Vis spectrophotometer. 6. Plasmids pAux/PylCUA or pAux/PylUACU/TyrCUA for double incorporation [20] (Table 1). 7. SS320 F′ electrocompetent cells bearing pAux/PylCUA (or pAux/PylUACU/TyrCUA for double incorporation). 8. E. coli SS320. 9. CM13 helper phage (Antibody Design Labs). 10. Isopropyl β-D-1-thiogalactopyranoside (IPTG). 11. ncAAs: We use CypK and PrpF for double incorporation (see Note 2). 12. Polyethylene glycol (PEG)/NaCl solution: 20% PEG-6000 (w/v), 2.5 M NaCl. Stored at 4 °C.

2.3 ELISA for Functional Analysis of Displayed scFv

1. Plate reader. 2. 96-well ELISA plates. 3. HER2 ectodomain (e.g., Sino Biologicals, 10,001-HCCH). 4. Coating buffer: Na2HCO3 100 mM, pH 9.4. 5. Phosphate-buffered saline (PBS). 6. PBS with 0.05% Tween-20 (PBST). 7. Bovine serum albumin (BSA). 8. Anti-M13-pVIII–HRP conjugate antibody (GE Healthcare Life Sciences 27-9421-01). 9. 3,3′,5,5′-Tetramethylbenzidine (TMB). 10. 1 M sulfuric acid.

2.4 Western Blot Analysis and Quantification

1. SDS-PAGE electrophoresis equipment. 2. β-Mercaptoethanol. 3. 4–12% Bis–Tris NuPAGE gels. 4. Western blot apparatus. 5. Fluorescence western blot imager (e.g., LI-COR Odyssey Blot Imager). 6. Loading buffer. 7. A transfer device for transferring proteins from PAGE to membrane. 8. PVDF membrane.

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9. Blocking buffer. 10. Anti-p3 (PSKAN3 MoBiTech). 11. 800CW streptavidin (926-32230 LI-COR). 12. Goat anti-mouse secondary antibody (680RD LI-COR). 13. Gel imager. 14. Densitometry analysis software (e.g., Fiji). 2.5 ncAA Conjugation Reactions

1. 400 μM Cy5 tetrazine in DMSO (CLK-015 Jena Bioscience). 2. 4 mM BCN-OH in DMSO. 3. Cy7 azide (CLJ-1052 Jena Bioscience). 4. CuSO4. 5. Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA). 6. Sodium ascorbate.

3

Methods

3.1 Cloning of the Target scFv into the Phagemid

1. Order the gene block encoding the target scFv with 5′ NcoI and 3′ SacI sites (see Note 1 and Table 1). 2. Digest 100 ng of the gene block with NcoI and SacI. After digestion, heat inactivate the endonucleases at 80 °C for 20 min. Purify the digested fragment using a DNA purification kit. 3. Digest 1 μg of O-scFv–g3 phagemid with NcoI and SacI. After digestion, heat inactivate the endonucleases at 80 °C for 20 min. Purify the digested fragment by gel electrophoresis and obtain the fragment using a gel extraction kit. 4. Ligate 50 ng of the digested gene block and 50 ng of digested O-ScFv–g3 phagemid backbone overnight at 16 °C. 5. Heat inactivate at 65 °C for 10 min and transform the ligation mixture. Allow to recover and plate cells on an LB agar plate with 50 μg/mL Amp. 6. Inoculate cells in 2xYT medium with 50 μg/mL Amp. 7. Extract the plasmid DNA on the following day and verify by Sanger sequencing.

3.2 Phage Expression with ncAAs

First, prepare electrocompetent SS320 F′ cells bearing pAux/PylCUA (or pAux/PylUACU/TyrCUA for double incorporation). 1. Inoculate 15 mL of 2xYT with one colony of SS320 F′ E. coli and incubate overnight at 37 °C with shaking. 2. Dilute 1:100 in 1 L final volume and grow at 37 °C with shaking until the OD600 reaches 0.4–0.6.

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3. Cool down on ice for 20 min with occasional gentle swirling. 4. Centrifuge at 5000 g at 4 °C for 10 min and resuspend the pellet. Repeat three times, each time decreasing amounts of ice-cold water. Use 800 mL, 400 mL, and 200 mL of 10% glycerol for the first, second, and third wash. Finally, resuspend cells in 2.5 mL of 10% glycerol with 150 mM trehalose. 5. Aliquot 55 μL in microcentrifuge tubes, flash freeze, and store at -80 °C. Then, proceed with the transformation steps. 6. Transform 10 ng of the ligated phagemid to the previously prepared SS320 cells by electroporation. 7. Plate cells on LB agar supplemented with 50 μg/mL Amp, 5 μg/mL Tet, and 37.5 μg/mL Spec and incubate overnight at 37 °C. 8. Inoculate 5–10 colonies in a shaking flask with 5 mL of 2xYT medium containing 50 μg/mL Amp, 5 μg/mL Tet, and 37.5 μg/mL Spec. All media should be supplemented with 2% glucose in order to inhibit the lacI promoter regulating the expression of the orthogonal ribosome. Incubate at 37 °C and at 250 rpm overnight. 9. Prepare 5 mL of culture by diluting the overnight culture to OD600 0.1–0.25 and grow to OD600 0.5–0.6 in 2xYT medium with the aforementioned antibiotics without glucose (see Note 3). 10. Add 5 μL of CM13 helper phage commercial stock to superinfect each sample. A multiplicity of infection (MOI) of 14 is used as recommended by the provider. Allow infection to proceed for 1 h at 37 °C and at 250 rpm. 11. Centrifuge the cells, discard the supernatant, and resuspend them in medium containing 50 μg/mL Kan, 50 μg/mL Amp, and 37.5 μg/mL Spec and IPTG 1 mM. 12. Add CypK or PrpF at a final concentration of 2 mM. Use 5 mM for double incorporation with PrpF to maximize expression efficiency. Enable expression at 30 °C and 250 rpm for 18 h (see Note 2). 13. Centrifuge cells at 3900 g for 10 min. Purify phage by two PEG/NaCl precipitation steps. Briefly, add 4 mL of supernatant containing phage to 1 mL of cooled PEG/NaCl solution and vortex. Incubate 1 h on ice. Centrifuge the samples at 3900 g for 45 min at 4 °C. Discard the supernatant and add 800 μL of PBS pipetting the solution 7–10 times. Add the 800 μL of PBS on 200 μL of ice-cold PEG/NaCl and allow to stand for 1 h. Then, centrifuge for 20 min at 3900 g and remove all supernatant by pipetting twice. Resuspend in 200 μL of PBS.

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3.3 Titration (See Note 4)

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1. For each sample, add 198 μL of SS320 F′ cells at OD600 = 0.5 to a 96-well plate (triplicates) and infect with 2 μL of phage concentrated 100-fold from the expression medium. 2. Place cells in a shaking incubator at 37 °C for 1 h and subsequently dilute in PBS tenfold eight times in a 96-well plate. 3. Spot 5 μL of each well containing diluted sample on a 9 cm LB– agar plate with 100 μg/mL Amp and allowed to grow at 30 °C for 16 h. 4. Calculate the concentration of phage from the number of colonies present on spots containing 3–30 colonies (see Note 5).

3.4 ELISA for Functional Analysis of Displayed scFv

1. Coat ELISA plates with 0.25 μg/mL of your antigen of choice in 100 μL of coating buffer (e.g., HER2 ectodomain in PBS overnight at 4 °C or for 2 h at room temperature). 2. Block plates with 1% BSA in PBS for 1 h at room temperature. 3. Wash wells with PBST for three times. 4. Dilute previously obtained phage samples 400 times and incubate 1–2 h at room temperature. 5. Wash the plate with PBST for five times. 6. Add 1:5000 anti-M13-pVIII–HRP conjugate antibody in 0.2% BSA in PBST for detection for 1 h at room temperature. 7. Wash wells with PBST four times and one last time with PBS. 8. Add TMB substrate and quench the reaction with 1 M sulfuric acid when sufficient color is developed (5–30 min). 9. Measure absorbance with microplate reader at 450 nm. After subtracting background absorbance at 570 nm, represent data in a standard curve fitted using a 5-parameter logistic regression (see Note 6).

3.5 Western Blot Analysis and Quantification

1. Dilute twofold 15 μL of samples from 50× stock and denature them for 20 min at 98 °C in NuPAGE LDS protein loading buffer supplemented with 10% β-mercaptoethanol. 2. Run the samples on a 4–12% Bis–Tris gel. 3. Transfer proteins from the gel to a PVDF membrane. 4. Block the membrane for 1 h using the blocking buffer. 5. Add anti-p3 and, if sample is biotinylated (see Note 7), 1:2000 800CW streptavidin (926-32230 LI-COR) 1:1000 overnight at 4 °C in PBS/blocking buffer with 0.2% Tween-20 and 0.01% SDS. 6. Wash with PBST (3 × 5 min). 7. Probe the membrane with goat anti-mouse secondary antibody 1:15000 for 1 h at room temperature. 8. Wash with PBST (3 × 5 min) and rinse with water. 9. Image western blots and quantify through densitometric analysis (see Note 8 and Fig. 2).

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Fig. 2 Western blot of biotinylated phage shows specific labelling of scFv–p3 fusion. The p3 and p3 fusions are probed with an anti-p3 antibody. Phage with an amber codon encoded in the scFv–g3 fusion is grown with and without CypK. As expected, scFv–g3 is only expressed in the presence of CypK, while in the absence only the naked p3 from the helper phage is observed. In the presence of CypK the scFv–g3 fusion is observed at a similar level as the wild-type control, which does not contain the amber codon. All samples are also treated with a tetrazine-biotin derivative and subsequently probed with labelled streptavidin. As expected, only the scFv–p3 fusion bearing CypK can be detected since it is the only one to react with biotin 3.6 ncAA Conjugation Reactions with Fluorophores or Biotin (See Notes 7 and 9)

1. Phage is used as concentrated 50 times from the expression supernatant as described in Subheading 3.2 step 9 (0.1–10 pM) in PBS. 2. Add 1 μL of Cy5 tetrazine from a 400 μM DMSO stock (20 μM final) to 20 μL of phage. Allow the mixture to react for 12–16 h at 20–22 °C (see Note 10). 3. Quench the reactions with 0.5 μL of 4 mM BCN-OH (100 μM final). 4. If a second ncAA bearing a terminal alkyne has been encoded, an azide-bearing fluorophore such as Cy7 azide can be combined in a one-pot reaction. Prepare a 25x “click mixture” by mixing 1.5 μL of water, 1 μL of CuSO4 25 μM in water, 5 μL of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand 25 μM in water, and 2.5 μL of sodium ascorbate 25 μM freshly prepared. Add 1 μL of Cy7 azide from a 2 mM stock (100 μM

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final) to 20 μL of phage followed by 0.8 μL of “click mixture.” Allow the components to react for 12–16 h at 20–22 °C. 5. For biotin labelling, add biotin-PEG4-tetrazine (CP-6001 ConjuProbe) from a 0.5 mM DMSO stock (10 μM final) to 20 μL of phage. Allow the mixture to react for 12–16 h at 20–22 °C. After this time, quench the reaction with 0.5 μL of 4 mM BCN-OH (100 μM final). 3.7 In-Gel Fluorescence

1. Run samples labelled with Cy5 and/or Cy7 on 4–12% Bis–Tris gels as described in Subheading 3.5. 2. Visualize in a gel imager. Image gels containing samples conjugated with Cy5 tetrazine using an LD635 laser and Cy5 filter settings (λex = 635 nm, λem = 670 ± 15 nm). If a second fluorophore such as Cy7 azide is added to react with a second ncAA, use an LD785 laser and IRlong filter settings (λex = 785 nm, λem = 825 ± 15 nm).

4

Notes 1. The scFv will be cloned by restriction digest and ligation between the PelB signal peptide and the HA tag peptide that precedes gIII in the O-ScFv–g3 phagemid. Therefore, add two sequences flanking the scFv, each including an endonuclease cut site, the necessary nucleotides to preserve the sequence in frame, and six additional nucleotides to facilitate digestion. We suggest addition of an NcoI cut site (agccggc|catggcg) at the 5′ end and SacI cut site (a|gctcttagggc) at the 3′ end. The scFv sequence should also contain an amber codon (TAG) at the site for the ncAA. If two amino acids are to be encoded, a quadruplet codon such as AGTA should also be added. Different positions to encode the ncAA should be tested for each particular scFv and ncAA. All positions tested in our system yielded the target protein, although with substantially different yields [20]. In a 4D5 anti-HER2 scFv, 128 and 252 (EU numbering, Table 2) are two positions that enable high ncAA incorporation efficiency. If two ncAAs are to be encoded, we recommend using the combination 128TAG and 252AGTA. 2. Although CypK is the amino acid described here, the same protocol can be applied to other amino acids that are substrates of PylRS variants [23, 26]. 3. This protocol can be easily scaled up and used to encode ncAAs in phage display libraries to perform selections. 4. Virions may be quantified by UV using the following formula: (absorbance at 269 - absorbance at 320)*6·1016/(number of bases/virion). However, in our hands the number of virions is not a good indicator of infective phage or display of functional proteins.

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Table 2 Antibody sequence Antibodies

Sequence

Anti-HER2 scFv 4D5

DIQMTQSPSSLSASVGDRVT ITCRASQDVNTAVAWYQQKPG KAPKLLIYSASFLYSGVPSRFSGSRSG TDFTLTISSLQPEDFATYYCQQH YTTPPTFGQGTKVEIKRTVAG GGGSGGGGSGGGGSGGGGSEVQL VESGGGLVQPGGS LRLSCAASGFNIKDTYIHWV RQAPGKGLEWVARIYPTNGYTRYADSVK GRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYA MDYWGQGTLVTVSSX

Positions with highly efficient ncAA incorporation suggested in the protocol are underlined and highlighted in bold. C-terminal residue X is absent in the parent scFv

5. Titers of phage displaying the scFv and incorporating CypK using this system are close to 1010 cfu·mL-1. The helper phage used is key to obtain high yields. Some batch-to-batch variability may be expected. 6. Functional ELISA with HER2 shows comparable binding capacity (roughly 40%) of modified phage with respect to wild-type phage, as tested directly from the PEG/NaCl purified samples. 7. Biotin functionalization enables facile dual imaging of modified and unmodified p3–ScFv fusion on western blot (see Fig. 2). 8. Western blot enables estimation of the ratio of modified vs unmodified p3 protein. Typical ratios are around 1:10, which indicates mostly monovalent display of the scFv on phage (see Fig. 2). 9. This system enables incorporation of one or two ncAAs enabling mutually orthogonal reactions in a one-pot procedure. This can be easily visualized by labelling each amino acid with fluorophores with nonoverlapping spectra. For maximal conjugation yield, it is preferable to perform reactions sequentially. 10. CypK enables rapid modification of phage. Although 20 h ensures maximal modification, 2 h provides >90% labelling.

Acknowledgements We thank Dr. Jason Chin for plasmid materials. B.O.-S. and C.D.-P. hold “la Caixa” Junior Leader (ID 100010434) and MSCA-PF (HORIZON-MSCA-2021-PF-101063066) fellowships, respectively. Illustrations were created with BioRender.com.

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References 1. Smith GP, Petrenko VA (1997) Phage display. Chem Rev 97:391–410 2. 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 3. Sidhu SS, Geyer CR (2017) Phage display in biotechnology and drug discovery. CRC Press, Boca Raton 4. Sunderland KS, Yang M, Mao C (2017) Phageenabled nanomedicine: from probes to therapeutics in precision medicine. Angew Chem Int Ed 56:1964–1992 5. Saw PE, Song EW (2019) Phage display screening of therapeutic peptide for cancer targeting and therapy. Protein Cell 10:787–807 6. Sandman KE, Benner JS, Noren CJ (2000) Phage display of selenopeptides. J Am Chem Soc 122:960–961 7. Lim RK, Li N, Ramil CP, Lin Q (2014) Fast and sequence-specific palladium-mediated cross-coupling reaction identified from phage display. ACS Chem Biol 9:2139–2148 8. Heinis C, Rutherford T, Freund S, Winter G (2009) Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Nat Chem Biol 5:502–507 9. Heinis C, Winter G (2015) Encoded libraries of chemically modified peptides. Curr Opin Chem Biol 26:89–98 10. Chin JW (2017) Expanding and reprogramming the genetic code. Nature 550:53–60 11. Young DD, Schultz PG (2018) Playing with the molecules of life. ACS Chem Biol 13:854– 870 12. de la Torre D, Chin JW (2021) Reprogramming the genetic code. Nat Rev Genet 22:169– 184 13. Tian F, Tsao M-L, Schultz PG (2004) A phage display system with unnatural amino acids. J Am Chem Soc 126:15962–15963 14. Tsao ML, Tian F, Schultz PG (2005) Selective Staudinger modification of proteins containing p-azidophenylalanine. Chembiochem 6:2147– 2149 15. Liu CC, Mack AV, Tsao M-L, Mills JH, Lee HS, Choe H, Farzan M, Schultz PG, Smider VV (2008) Protein evolution with an expanded genetic code. Proc Natl Acad Sci U S A 105: 17688–17693

16. Liu CC, Mack AV, Brustad EM, Mills JH, Groff D, Smider VV, Schultz PG (2009) The evolution of proteins with genetically encoded “chemical warheads”. J Am Chem Soc 131: 9616–9617 17. Kang M, Light K, Ai HW, Shen W, Kim CH, Chen PR, Lee HS, Solomon EI, Schultz PG (2014) Evolution of iron(II)-finger peptides by using a bipyridyl amino acid. Chembiochem 15:822–825 18. Allen GL, Grahn AK, Kourentzi K, Willson RC, Waldrop S, Guo J, Kay BK (2022) Expanding the chemical diversity of M13 bacteriophage. Front Microbiol 13:961093 19. Owens AE, Iannuzzelli JA, Gu Y, Fasan R (2020) MOrPH-PhD: an integrated phage display platform for the discovery of functional genetically encoded peptide macrocycles. ACS Cent Sci 6:368–381 20. Oller-Salvia B, Chin JW (2019) Efficient phage display with multiple distinct non-canonical amino acids using orthogonal ribosomemediated genetic code expansion. Angew Chem Int Ed 58:10844–10848 21. Chen T, Hongdilokkul N, Liu Z, Adhikary R, Tsuen SS, Romesberg FE (2016) Evolution of thermophilic DNA polymerases for the recognition and amplification of C2′-modified DNA. Nat Chem 8:556–562 22. Chin JW (2014) Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83:379–408 23. Bryson DI, Fan C, Guo L-T, Miller C, So¨ll D, Liu DR (2017) Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol 13:1253 24. Wang XS, Chen P-HC, Hampton JT, Tharp JM, Reed CA, Das SK, Wang D-S, Hayatshahi HS, Shen Y, Liu J, Liu WR (2019) A genetically encoded, phage-displayed cyclic-peptide library. Angew Chem Int Ed 58(44): 15904–15909 25. Tharp JM, Hampton JT, Reed CA, Ehnbom A, Chen P-HC, Morse JS, Kurra Y, Pe´rez LM, Xu S, Liu WR (2020) An amber obligate active site-directed ligand evolution technique for phage display. Nat Commun 11:1392 26. Polycarpo CR, Herring S, Be´rube´ A, Wood JL, So¨ll D, Ambrogelly A (2006) Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett 580:6695–6700

Chapter 9 Genetically Encoded Noncanonical Amino Acids in Proteins to Investigate Lysine Benzoylation An-Di Guo and Xiao-Hua Chen Abstract Posttranslational modifications (PTMs) of lysine residues are major regulators of gene expression, protein– protein interactions, and protein localization and degradation. Histone lysine benzoylation is a recently identified epigenetic marker associated with active transcription, which has physiological relevance distinct from histone acetylation and can be regulated by debenzoylation of sirtuin 2 (SIRT2). Herein, we provide a protocol for the incorporation of benzoyllysine and fluorinated benzoyllysine into full-length histone proteins, which further serve as benzoylated histone probes with NMR or fluorescence signal for investigating the dynamics of SIRT2-mediated debenzoylation. Key words Non-canonical amino acid, Genetic code expansion, Lysine benzoylation, Histone benzoylation, Debenzoylation, 19F NMR spectroscopy, Fluorescence imaging

1

Introduction Lysine residues of proteins in cells undergo extensive posttranslational modifications (PTMs) that can regulate protein structure, function, and protein–protein interactions thereby affecting the biological process and fate of proteins [1–3]. Moreover, histone PTMs play important roles in the regulation of chromatin structure and function, thus serving as key epigenetic regulators involved in a range of biological processes including transcription, DNA repair, replication, and apoptosis [1–5]. Recently, a wide range of histone lysine acylations have been reported [4–7], of which histone lysine benzoylation (Kbz) as a newly identified epigenetic mark proved to correlate with active transcription and has physiological relevance distinct from histone lysine acetylation [7]. Very recently, human double PHD finger (DPF) and YEATS (Yaf9, ENL, AF9, Taf14, and Sas5) domain proteins have been identified as the first readers for histone benzoylation according to the systematic binding studies and structural analyses, suggesting

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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lysine benzoylation and its readout as an important mechanism for gene regulation [8]. In addition, several studies have reported that sodium benzoate exhibits various biological activities, such as antiinflammatory and immunomodulatory activities, and neuroprotective function, as well as potential therapeutic applications in multiple sclerosis and cancer [9–12]. Nevertheless, other studies have revealed that sodium benzoate, also a popular food additive agent, can induce developmental defects, oxidative stress, and anxiety-like behavior in zebrafish larva and impaired memory performance and increased brain oxidative stress in mice and is associated with increased reporting of attention-deficit hyperactivity disorder symptoms (ADHD) in humans [13–15]. Clearly, the discovery of histone benzoylation and its regulatory mechanisms opens up a new and important avenue to investigate the histone epigenetics, biological functions, and cellular impact of the sodium benzoate treatment, thus for better understanding of its downstream effects in physiological processes and pathophysiology [8, 16–19]. The site-specific introduction of modified lysine residues into proteins can be achieved by genetic code expansion technology, conferring unique advantages of site specificity and full-length proteins for interrogating PTMs in vitro and in living cells, which serves as an efficient chemical biology tool for investigating the reversible process of PTMs and the molecular mechanisms [20– 25]. Various histone lysine acylations have been studied via genetic code expansion, including acetylation (Kac) [26, 27], crotonyllysine (Kcr) [28, 29], 2-hydroxyisobutyryllysine (Khib) [30], propionyllysine (Kpr) [31], butyryllysine (Kbu) [32], and others [33– 37]. We recently demonstrated the genetic incorporation of benzoyllysine and fluorinated benzoyllysine into full-length histone proteins with site-specific manner in live cells, based on our rational-designed synthetase and fine-integrated fluorine element into benzoyllysine (Fig. 1) [38]. We describe here a general procedure for incorporating benzoyllysine and fluorinated benzoyllysine (Fig. 1) into proteins in E. coli and mammalian cells. The site-specific incorporated noncanonical amino acids (ncAAs) are versatile probes for investigating histone benzoylation under biological environments, conferring multiplex signals such as 19F NMR spectra with chemical clarity and fluorescent signal for benzoylation. In addition, the sitespecific incorporated lysine benzoylation within full-length histone proteins revealed distinct dynamics of debenzoylation in the presence of the SIRT2 [38].

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Fig. 1 Site-specific and genetic encoding of benzoyllysine (Kbz) and fluorinated benzoyllysines (F-Kbz and 2F-Kbz) into full-length histones in living cell to investigate histone benzoylation and benzoylation–enzyme interactions via the genetic code expansion strategy

2

Materials Unless otherwise stated, all solutions are prepared and stored at room temperature using Ultrapure water (e.g., Milli-Q water) and analytical grade reagents.

2.1 Cell Lines, Plasmids, and ncAAs

1. E. coli BL21(DE3). 2. E. coli DH10B. 3. HEK293T cell line. 4. A plasmid containing the ORF of the protein of interest with an amber codon at the desired position for ncAA (e.g., pBAD-Ub3TAG, pTAK-H3–9TAG, or pcDNA3.1-H2B-16TAG in Fig. 2; see Note 1). 5. A plasmid expressing the orthogonal aaRS/tRNA for ncAA incorporation (e.g., pEvol-MmPylRS(Y384F), pBK-MmPylRS (Y384F), or pNEU-MmPylRS(Y384F) in Fig. 2; see Note 2). 6. Plasmid pET28a-SIRT2 for expression of wild-type SIRT2 (see Note 3). 7. ncAA Kbz, F-Kbz, and 2F-Kbz (Fig. 1) are synthesized from commercially available materials in two steps (see Note 4).

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Fig. 2 Representative plasmid maps. (a) The maps of plasmids used for Ub-3TAG expression in E. coli, including the plasmid of POI (protein of interest) pBAD-Ub-3TAG and aminoacyl-tRNA synthetase plasmid

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1. 2YT medium: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.4. This solution should be autoclaved before use and used within 2 days (see Note 5). 2. SOC medium: 20 g/L tryptone, 5 g/L yeast extract, 10 mM NaCl, and 25 mM KCl. Add MgCl2 to 10 mM and glucose to 20 mM after autoclaving (see Note 5). 3. 1000× kanamycin sulfate stock solution: Dissolve 500 mg kanamycin sulfate in 10 mL water. This solution should be sterile filtered, aliquoted, and stored at -20 °C. 4. 1000× chloramphenicol stock solution: Dissolve 340 mg chloramphenicol sulfate in 10 mL ethanol. This solution should be sterile filtered, aliquoted, and stored at -20 °C 5. 1000× ampicillin stock solution: Dissolve 1 g ampicillin in 10 mL water. This solution should be sterile filtered, aliquoted, and stored at -20 °C. 6. 1000× IPTG stock solution: Dissolve 2.38 g IPTG in 10 mL water to 1 M. This solution should be sterile filtered, aliquoted, and stored at -20 °C. 7. 100× arabinose stock solution: Dissolve 20 g arabinose in 80 mL water to 20% (w/w). This solution should be sterile filtered, aliquoted, and stored at -20 °C. 8. Agarose 2YT plate: Prepare 2YT medium and add 20 g/L agarose. Autoclave at 121 °C for 15 min. Let the medium cool to ~55 °C, and then add corresponding antibiotics. Mix completely and pour plates. Allow the medium to solidify at room temperature. The plates can be stored at 4 °C for a month and should be pre-warmed at 37 °C before use. 9. Native lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% v/v glycerol, 1% v/v Tween 20, 0.5 mg/mL lysozyme, 1 mM PMSF, pH 7.4. This solution should be sterile filtered and stored at 4 °C. 10. Ni-NTA native wash buffer: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.4. This solution should be sterile filtered and stored at 4 °C.

ä Fig. 2 (continued) pEvol-MmPylRS(Y384F). The pEvol plasmid contains synthetase MmPylRS(Y384F) and tRNApylCUA. (b) The maps of plasmids used for H3-9TAG expression in E. coli, including plasmid of POI pTAKH3-9TAG and aminoacyl-tRNA synthetase plasmid pBK-MmPylRS(Y384F). The pBK plasmid contains synthetase MmPylRS(Y384F) and the pTAK plasmid contains tRNApylCUA. (c) The maps of plasmids used for H2B-16TAG-mKate2 expression in mammalian cells, including plasmid of POI pcDNA3.1-H2B-16TAGmKate2 and aminoacyl-tRNA synthetase plasmid pNEU-MmPylRS(Y384F). The pNEU plasmid contains one copy of synthetase MmPylRS(Y384F) and four copies of tRNA U6M15

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11. Ni-NTA native elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 7.4. This solution should be sterile filtered and stored at 4 °C. 12. Denaturing lysis buffer: 50 mM Tris–HCl, 500 mM NaCl, 6 M urea, pH 8.0. This solution should be sterile filtered. 13. Ni-NTA denaturing wash buffer: 50 mM Tris–HCl, 500 mM NaCl, 6 M urea, 20 mM imidazole, pH 8.0. This solution should be sterile filtered. 14. Ni-NTA denaturing elution buffer: 50 mM Tris–HCl, 500 mM NaCl, 6 M urea, 250 mM imidazole, pH 8.0. This solution should be sterile filtered. 15. Dialysis buffer with a linear gradient of urea from 6 M to 0: 50 mM Tris–HCl, 500 mM NaCl, 6/5/4/3/2/1/0 M urea, pH 8.0. This solution should be sterile filtered and pre-cooled at 4 °C before used. 16. PBS: 1.06 mM KH2PO4, 155.17 mM NaCl, and 2.97 mM Na2HPO4.7H2O, pH 7.4 (used for protein preservation). This solution should be sterile filtered and pre-cooled at 4 °C if needed. 17. Ni-NTA slurry (Qiagen). The slurry should be stored at 4 °C. 18. 4× Laemmli buffer (Bio-Rad). The buffer should be added DTT (8 mM) before use. 19. PAGE gels: 15% acryl/bis (29:1), 7 cm × 8 cm, 1 mm thick, Tris–HCl (see Note 6). 20. SDS-PAGE running buffer: 50 mM Tris–HCl, 384 mM glycine, 0.1% SDS, pH 8.3 (see Note 7). 21. Coomassie staining solution: 0.1% (w/v) Coomassie Brilliant Blue R250, 30% (v/v) ethanol, 10% (v/v) acetic acid in water (see Note 8). 22. Destaining solution: 30% (v/v) ethanol, 10% (v/v) acetic acid in water. 23. Buffer (20 mM Tris–HCl, 300 mM NaCl, 1 mM DTT, pH = 7.4): The buffer should be sterile filtered and stored at room temperature within 2 days. 2.3

19

F NMR

1. NMR buffer: 10% (v/v) D2O, 20 mM Tris–HCl, 300 mM NaCl, 1 mM DTT, pH 7.4. 2. NMR spectrometer (e.g., Bruker Avance III HD 500 MHz NMR using a BBFO Smart 5 mm probe).

2.4

Debenzoylation

1. Reaction buffer: 20 mM Tris–HCl, 1 mM DTT, 1 mM NAD+, 100 mM NaCl, pH 8. 2. 1.6 M acetic acid in methanol.

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3. ESI-TOF LC-MS with a C4 column (e.g., Waters ACQUITY UPLC Protein BEH C4, 1.7 μm, 2.1 mm x 50 mm). 2.5 Transfection and Imaging

1. DMEM medium. 2. FBS. 3. Pen-Strep (e.g., Gibco). 4. Opti-MEM medium (e.g., Gibco). 5. Transfection reagent (e.g., Invitrogen Lipofectamine 3000). 6. Fixing solution: 4% PFA in PBS. 7. Precoated cell culture coverslips: Disperse the coverslip in 0.1 mg/mL poly-D-lysine and incubate at room temperature overnight. Remove solution and dry the coverslip at room temperature overnight. 8. Confocal microscope (e.g., Leica TCS-SP8 STED).

3

Methods

3.1 Plasmid Preparation

1. Acquire a pBAD, pTAK, or pcDNA3.1 vector containing the ORF of the protein of interest (POI). 2. To your gene of interest in pBAD or pTAK or pcDNA3.1 vector add an amber codon (TAG) in the location that an ncAA is desired (Tables 1 and 2) (see Note 9). Select individual colonies to verify mutations by DNA sequencing.

3.2 Protein Expression 3.2.1 Expression and Purification of KbzContaining Ubiquitin Under Native Conditions

1. Add plasmids pBAD-Ub-3TAG and pEvol-MmPylRS(Y384F) to 100 μL of DH10B competent cells as in Table 3. Incubate on ice for 20 min. Place the tubes for 45 s at 42 °C and leave them on ice for 2 min. Add 1 mL of pre-warmed SOC medium to the tube and incubate at 37 °C for 1 h at 250 rpm. Spread 50 μL of mixture of cells on a pre-warmed 2YT agarose plate containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol. Incubate the plates overnight at 37 °C. 2. Pick a single colony from the 2YT agarose plate into 5 mL of 2YT medium containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol. Incubate in a shaker at 37 °C, 180 rpm, overnight. 3. Measure the OD600 of the culture, and inoculate into 200 mL of fresh 2YT medium containing corresponding antibiotics. Culture the cells at 37 °C, 200–250 rpm until OD600 ~0.5 is reached. Divide the cultured cells into two 100 mL portions as experimental group added ncAA and control group without ncAA. Add the corresponding ncAA (F-Kbz or 2F-Kbz) as in Table 3 to a final concentration of 1 mM to the experimental group. Add the same volume of medium to the control group. Culture the cells at 37 °C, 200–250 rpm, for

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Table 1 Protein sequences of POI incorporated with ncAAs Kbz, F-Kbz, or 2F-Kbz and wild-type SIRT2 Protein

Protein sequence

Ub-3TAG

MTSM*IFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQ LEDGRTLSDYNIQKESTLHLVLRLRGLEHHHHHHHH (* = F-Kbz or 2F-Kbz)

H3-9TAG

MTSARTKQTAR*STGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALR EIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLV GLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERARSHHHHHH (* = 2FKbz)

H3-18TAG

MTSARTKQTARKSTGGKAPR*QLATKAARKSAPATGGVKKPHRYRPGTVALR EIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLV GLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERARSHHHHHH (* = Kbz)

H3-27TAG

MTSARTKQTARKSTGGKAPRKQLATKAAR*SAPATGGVKKPHRYRPGTVAL REIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAY LVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERARSHHHHHH (* = Kbz)

H2B-16TAGmKate2

MPEPAKSAPAPKKGSK*AVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDT GISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGE LAKHAVSEGTKAITKYTSAKDPPVATMVSELIKENMHMKLYMEGTVNNH HFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHT QGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGV NFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICN LKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCD LPSKLGHRTGHHHHHH (* = Kbz or 2F-Kbz)

H2B-20TAGmKate2

MPEPAKSAPAPKKGSKKAVT*AQKKGGKKRKRSRKESYSIYVYKVLKQVHPDT GISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGE LAKHAVSEGTKAITKYTSAKDPPVATMVSELIKENMHMKLYMEGTVNNH HFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINH TQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIR GVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHL ICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY CDLPSKLGHRTGHHHHHH (* = Kbz or 2F-Kbz)

Wild-type SIRT2

MGMHHHHHHGSDFLRNLFSQTLSLGSQKERLLDELTLEGVARYMQSERC RRVICLVGAGISTSAGIPDFRSPSTGLYDNLEKYHLPYPEAIFEISYFKKHPE PFFALAKELYPGQFKPTICHYFMRLLKDKGLLLRCYTQNIDTLERIAGLEQ EDLVEAHGTFYTSHCVSASCRHEYPLSWMKEKIFSEVTPKCEDCQSLVKP DIVFFGESLPARFFSCMQSDFLKVDLLLVMGTSLQVQPFASLISKAPLSTPR LLINKEKAGQSDPFLGMIMGLGGGMDFDSKKAYRDVAWLGECDQGCLA LAELLGWKKELEDLVRREHASIDAQSGAGVPNPSTSASPKKSPPPAKDEAR TTEREKPQ

another 10 min. Change the culturing temperature to 30 °C and culture at 250 rpm for 20 min. Add arabinose to a final concentration of 0.2% to both groups. Culture the induced cells for 12 h at 30 °C, 250 rpm.

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Table 2 Plasmids used for POI expression in different cells

Protein

Plasmid of POI

Aminoacyl-tRNA synthetase plasmid

Cell

Ub-3TAG

pBAD-Ub-3TAG

pEvol-MmPylRS(Y384F)

E. coli DH10B

H3-9TAG

pTAK-H3-9TAG

pBK-MmPylRS(Y384F)

E. coli BL21 (DE3)

H3-18TAG

pBAD-H3-18TAG

pEvol-MmPylRS(Y384F)

E. coli DH10B

H3-27TAG

pBAD-H3-27TAG

pEvol-MmPylRS(Y384F)

E. coli DH10B

H2B-16TAGmKate2

pcDNA3.1-H2B-16TAGmKate2

pNEU-MmPylRS(Y384F)

HEK293T

H2B-20TAGmKate2

pcDNA3.1-H2B-20TAGmKate2

pNEU-MmPylRS(Y384F)

HEK293T

Table 3 Expression conditions of ubiquitin with a ncAA at position 3 Protein

Ub-3(F-Kbz)

Ub-3(2F-Kbz)

Ubiquitin plasmid

pBAD-Ub-3TAG

pBAD-Ub-3TAG

Aminoacyl-tRNA synthetase plasmid

pEvol-MmPylRS(Y384F)

pEvol-MmPylRS(Y384F)

ncAA

F-Kbz (1 mM)

2F-Kbz (1 mM)

4. Collect cells of experimental and control groups. Centrifuge cells at 4200 g for 20 min at 4 °C and store at -80 °C overnight. 5. Resuspend the cell pellets of experimental and control groups in 5 mL native lysis buffer. Lyse the cells with ultrasonication for 15 min (40% power, 2 s on, 4 s off, 10 min). Centrifuge the lysate at 15,000 g for 30 min at 4 °C. Incubate the supernatants with pre-equilibrated 100 μL of Ni-NTA agarose resin at 4 °C for 2 h with gentle shaking. 6. Load the slurry onto a chromatography column and wash with 3 mL of Ni-NTA native wash buffer. Elute the His tag proteins with 100 μL of Ni-NTA native elution buffer for 10 times. Monitor the content and purity of the eluted protein by SDS-PAGE and Coomassie staining. 7. Dialyze pure proteins to the corresponding buffer for analysis or store.

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3.2.2 Expression and Purification of KbzContaining Histone H3 Under Denaturing Conditions

1. Add plasmids of H3-TAG mutant and corresponding aminoacyl tRNA synthetase plasmids to 100 μL of corresponding E. coli competent cells as in Table 4. Incubate on ice for 20 min. Place the tubes for 45 s at 42 °C and leave them on ice for 2 min. Add 1 mL of pre-warmed SOC medium to the tube and incubate at 37 °C for 1 h at 250 rpm. Spread 50 μL of mixture of cells on a pre-warmed 2YT agarose plate with corresponding antibiotics as in Table 4. Incubate the plates overnight at 37 °C. 2. Pick a single colony from the 2YT agarose plate, suspend in 5 mL 2Y of T medium containing corresponding antibiotics, and vortex to disperse. Incubate in a shaker at 37 °C and 180 rpm overnight. 3. Measure the OD600 of the culture, and inoculate into 200 mL of fresh 2YT medium containing corresponding antibiotics. Culture the cells at 37 °C and 200–250 rpm until OD600 ~0.3 is reached, and add nicotinamide to a final concentration of 20 mM. Culture the cells at 37 °C and 200–250 rpm until OD600 ~0.5 is reached. Divide the cultured cells into two 100 mL portions as the experimental and control groups. Add the corresponding ncAA as in Table 4 to the experimental group. Add the same volume of medium to the control group. Culture the cells at 37 °C and 200–250 rpm for another 30 min. Add corresponding inducer as in Table 4 to both groups. Culture the induced cells for 12 h at 37 °C and 250 rpm.

Table 4 Expression conditions of histone H3 with a ncAA at position 9, 18, or 27 Protein

H3-9(2F-Kbz)

H3-18Kbz

H3-27Kbz

H3 plasmid and pTAK-H3-9TAG TAG site

pBAD-H3-18TAG

pBAD-H3-27TAG

AminoacyltRNA synthetase plasmid

pBK-MmPylRS(Y384F)

pEvol-MmPylRS(Y384F)

pEvol-MmPylRS(Y384F)

Cell

BL21(DE3)

DH10B

DH10B

Antibiotics

50 μg/mL kanamycin sulfate and 34 μg/mL chloramphenicol

100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol

100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol

ncAA

2F-Kbz (1 mM)

Kbz (3 mM)

Kbz (3 mM)

Inducer

IPTG (1 mM)

Arabinose (0.2%)

Arabinose (0.2%)

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4. Collect cells of experimental and control groups, respectively. Centrifuge cells at 4200 g for 20 min at 4 °C and store at -80 ° C overnight. 5. Resuspend the cell pellets of the experimental and control groups separately in 10 mL of 50 mM Tris–HCl buffer (pH 8.0) containing 500 mM NaCl. Lyse the cells with ultrasonication for 15 min (40% power, 2 s on, 4 s off, 10 min). Centrifuge the lysate at 12,000 g for 30 min at 4 °C. Discard the supernatants, suspend the cell pellets in 10 mL of 2% Triton X-100, incubate on ice for 10 min, and centrifuge. Repeat the wash of inclusion body pellets with 10 mL of 2% Triton X-100. Wash the inclusion body pellets with 10 mL of 50 mM Tris– HCl buffer (pH 8.0) containing 500 mM NaCl. Resuspend the inclusion body pellets with 5 mL of denaturing lysis buffer containing 1 mM DTT. Dissolve the inclusion body pellets with ultrasonication (40% power, 2 s on, 4 s off, 10 min). Centrifuge the lysate at 12,000 g for 30 min at 25 °C. Incubate the supernatants with pre-equilibrated 100 μL of Ni-NTA agarose resin at 25 °C for 2 h with gentle shaking. 6. Load the Ni-NTA slurry onto a chromatography column and wash with 3 mL of Ni-NTA denaturing wash buffer. Elute the His tag proteins with 100 μL of Ni-NTA denaturing elution buffer for 10 times. Monitor the content and purity of the eluted protein by SDS-PAGE and Coomassie staining. 7. Renature the purified protein via dialysis with a linear gradient of urea from 6 M to 0; finally exchange the protein to the corresponding buffer for analysis or storage. 3.2.3 Expression and Purification of Wild-Type SIRT2 Protein

1. Add plasmid pET28a-SIRT2 to 100 μL of BL21(DE3) competent cells. Incubate on ice for 20 min. Place the tubes for 45 s at 42 °C and leave them on ice for 2 min. Add 1 mL of pre-warmed SOC medium to the tube and incubate at 37 °C for 1 h at 250 rpm. Spread 50 μL of mixture of cells on a pre-warmed 2YT agarose plate containing 50 μg/mL of kanamycin sulfate. Incubate the plates overnight at 37 °C. 2. Pick a single colony from the 2YT agarose plate, suspend in 5 mL of 2YT medium containing corresponding antibiotics, and vortex to disperse. Incubate in a shaker at 37 °C and 180 rpm overnight. 3. Measure the OD600 of the culture, and inoculate into 100 mL of fresh 2YT medium containing corresponding antibiotics. Culture the cells at 37 °C, 250 rpm, until OD600 ~ 0.8 is reached. Add IPTG to a final concentration of 0.5 mM. Culture the induced cells for 2 h at 37 °C and 250 rpm. 4. Collect cells and centrifuge cells at 4200 g for 20 min at 4 °C and store at -80 °C overnight.

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5. Resuspend the cell pellets in 5 mL of nondenaturing lysis buffer. Lyse the cells with ultrasonication for 15 min (40% power, 2 s on, 4 s off, 10 min). Centrifuge the lysate at 15,000 g for 30 min at 4 °C. Incubate the supernatants with pre-equilibrated 100 μL of Ni-NTA agarose resin at 4 °C for 2 h with gentle shaking. 6. Load the slurry onto a chromatography column and wash with 3 mL of Ni-NTA native wash buffer. Elute the His tag proteins with 100 μL of Ni-NTA native elution buffer for 10 times. Monitor the content and purity of the eluted protein by SDS-PAGE and Coomassie staining. 7. Dialyze pure proteins to the corresponding buffer for analysis or store. 3.3 Debenzoylation of Modified Histone H3 by SIRT2

1. Add histone H3-18Kbz or H3-27Kbz (4 μM) and SIRT2 (0.2 μM) to 120 μL of reaction buffer and mix well. Divide the mixture into six parts equally, and incubate them at 37 °C for 0/10/20/40/80/160 min, respectively (see Note 10). 2. Stop the debenzoylation reaction by adding 4 μL of 1 M HCl and 1.6 M acetic acid in methanol. Analyze the samples by ESI-TOF LC-MS at 80 °C (see Note 11). 3. Quantify the debenzoylated product by their peak areas.

3.4 19F NMR Experiments for Proteins with Fluorinated Benzoyllysine

1. Prepare ubiquitin protein, or histone H3 protein sample in NMR buffer 10% D2O.

3.5 Transfection and Imaging of H2BmKate2 Protein Containing a Kbz

1. Place precoated cell culture coverslips into each well of 12-well plates. Inoculate 1.0 × 105 of HEK293 T cells in the well, containing 1 mL of DMEM medium supplemented with 10% FBS and 1% Pen-Strep. Incubate the cells at 37 °C in an incubator with a 5% CO2 atmosphere. The cell confluency will reach ~60% after 16–18 h of incubation.

2. Perform 19F NMR experiments on an NMR spectrometer. 3. Acquire 19F NMR spectra with 1-s delay at 298 K.

2. Replace the growth medium with 1 mL of fresh medium containing 1 mM Kbz, 2 mM 2F-Kbz, or equal volume of DMSO as negative control and incubate for 30 min. 3. Mix 50 μL of Opti-MEM with 1 μg of the histone plasmid carrying the amber mutant gene, together with 1 μg of the aminoacyl-tRNA synthetase plasmid as in Table 5. Add 4 μL of P3000 and mix well. Incubate the mixture at room temperature for 5 min.

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Table 5 Transfection conditions of histone H2B with an ncAA at position 16 or 20 Protein

H2B-16TAG-mKate2

H2B-20TAG-mKate2

Histone plasmid and TAG site

pcDNA3.1-H2B 16TAGmKate2

pcDNA3.1-H2B 20TAGmKate2

Aminoacyl-tRNA synthetase plasmid

pNEU-MmPylRS(Y384F)

pNEU-MmPylRS(Y384F)

ncAA

Kbz (1 mM) or 2F-Kbz (2 mM) Kbz (1 mM) or 2F-Kbz (2 mM)

4. In parallel with step 3, mix 6 μL of Lipofectamine 3000 with 50 μL of Opti-MEM, and then incubate the mixture at room temperature for 5 min. 5. Combine these mixtures, prepared at steps 3 and 4, in a tube and mix them gently. Incubate the combined mixture at room temperature for 15 min. 6. Add the transfection mixture to the well and mix gently by rocking the culture plate. Incubate the cells at 37 °C in the 5% CO2 incubator for 48 h. 7. Remove the medium and wash cells gently with PBS (1 mL × 2). Add 1 mL 4% PFA in PBS and incubate for 10 min at room temperature. Remove PFA solution and wash cells gently with PBS (1 mL × 2) with 5-min terminal. Collect the coverslips and mount each coverslip with a drop of mounting medium that provides additional protection from bleaching. Observe the cells under a confocal microscope.

4

Notes 1. The expression plasmid and aminoacyl-tRNA synthetase plasmid will be co-transformed into an expression E. coli strain. Therefore, any expression vector used for the protein of interest should have an alternative selection marker and origin of replication. 2. The plasmid pNEU-MmPylRS(Y384F) can be constructed from the pNEU-hMbPylRS-4xU6M15 (Addgene, #105830) backbone by substituting the sequence of hMbPylRS with MmPylRS(Y384F) [39]. The plasmid pEvol-MmPylRS (Y384F) can be constructed from the pEvol-pAzF (Addgene, #31186) backbone by substituting the two copies of pAzF with one copy of MmPylRS(Y384F) and the tRNA sequence with the PylT sequence as gggaacctgatcatgtagatcgaatggactctaaatcc gttcagccgggttagattcccggggtttccg [39].

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3. We purchased pET28a-SIRT2 from custom DNA synthesis with SIRT2 inserted between NcoI and NotI sites. The protein sequence of wild-type SIRT2 is provided in Table 1. 4. Kbz, F-Kbz, and 2F-Kbz are synthesized following the reported procedures [38]. 5. The medium should be autoclaved, cooled down to the room temperature, and the corresponding antibiotics added before use. Relevant experiments should follow aseptic technique. 6. The gel can easily be adjusted for appropriate resolution of the protein of interest. 7. The SDS-PAGE running buffer was prepared by diluting 10× buffer. 8. The Coomassie staining solution was prepared by first dissolving Coomassie Brilliant Blue R250 in ethanol and acetic acid and then diluting with water. 9. The plasmids containing the wild-type DNA sequence can be purchased from companies providing DNA gene synthesis service. The mutation can be introduced by overlapping PCR and recombinant cloning. 10. Incubation should be conducted in a thermo shaker at 37 °C and 200 rpm. 11. For protein mass analysis in SIRT2-catalyzed debenzoylation, the injection of 1 μg protein is enough. The sample can be analyzed using 5–95% acetonitrile gradient (containing 0.1% formic acid) for 10 min.

Acknowledgments Plasmids pTAK and pBK-MmPylRS were gifts from Prof. Lei Wang, plasmid pEvol was a gift from Prof. Peter Schultz (Addgene plasmid 31186), and plasmid pNEU-hMbPylRS-4xU6M15 was a gift from Prof. Irene Coin (Addgene plasmid # 105830). References 1. Sabari BR, Zhang D, Allis CD, Zhao YM (2017) Metabolic regulation of gene expression through histone acylations. Nat Rev Mol Cell Bio 18(2):90–101 2. Zhang ZH, Tan MJ, Xie ZY, Dai LZ, Chen Y, Zhao YM (2011) Identification of lysine succinylation as a new post-translational modification. Nat Chem Biol 7(1):58–63 3. Dai LZ, Peng C, Montellier E, Lu ZK, Chen Y, Ishii H, Debernardi A, Buchou T, Rousseaux S, Jin FL, Sabari BR, Deng ZY, Allis CD, Ren B,

Khochbin S, Zhao YM (2014) Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark. Nat Chem Biol 10(5):365–373 4. Huang H, Sabari BR, Garcia BA, Allis CD, Zhao YM (2014) SnapShot: histone modifications. Cell 159(2):458 5. Suganuma T, Workman JL (2011) Signals and combinatorial functions of histone modifications. Annu Rev Biochem 80:473–499

Genetically Encoded Benzoyllysines in Proteins 6. Zhang D, Tang ZY, Huang H, Zhou GL, Cui C, Weng YJ, Liu WC, Kim S, Lee S, Perez-Neut M, Ding J, Czyz D, Hu R, Ye Z, He MM, Zheng YG, Shuman HA, Dai LZ, Ren B, Roeder RG, Becker L, Zhao YM (2019) Metabolic regulation of gene expreshistone lactylation. Nature sion by 574(7779):575 7. Huang H, Zhang D, Wang Y, Perez-Neut M, Han Z, Zheng YG, Hao Q, Zhao YM (2018) Lysine benzoylation is a histone mark regulated by SIRT2. Nat Commun 9:3374 8. Ren XL, Zhou Y, Xue ZY, Hao N, Li YY, Guo XH, Wang DL, Shi XB, Li HT (2021) Histone benzoylation serves as an epigenetic mark for DPF and YEATS family proteins. Nucleic Acids Res 49(1):114–126 9. Khasnavis S, Pahan K (2012) Sodium benzoate, a metabolite of cinnamon and a food additive, upregulates neuroprotective Parkinson disease protein DJ-1 in astrocytes and neurons. J Neuroimmune Pharm 7(2):424–435 10. Modi KM, Jana M, Mondal S, Pahan K (2015) Sodium benzoate, a metabolite of cinnamon and a food additive, upregulates ciliary neurotrophic factor in astrocytes and oligodendrocytes. Neurochem Res 40(11):2333–2347 11. Brahmachari S, Jana A, Pahan K (2009) Sodium benzoate, a metabolite of cinnamon and a food additive, reduces microglial and astroglial inflammatory responses. J Immunol 183(9):5917–5927 12. Yilmaz B, Karabay AZ (2018) Food additive sodium benzoate (NaB) activates NF kappa B and induces apoptosis in HCT116 cells. Molecules 23(4):723 13. Gaur H, Purushothaman S, Pullaguri N, Bhargava Y, Bhargava A (2018) Sodium benzoate induced developmental defects, oxidative stress and anxiety-like behaviour in zebrafish larva. Biochem Biophys Res Commun 502(3): 364–369 14. Khoshnoud MJ, Siavashpour A, Bakhshizadeh M, Rashedinia M (2018) Effects of sodium benzoate, a commonly used food preservative, on learning, memory, and oxidative stress in brain of mice. J Biochem Mol Toxic 32(2). https://doi.org/10.1002/jbt. 22022 15. Beezhold BL, Johnston CS, Nochta KA (2014) Sodium benzoate-rich beverage consumption is associated with increased reporting of ADHD symptoms in college students a pilot investigation. J Atten Disord 18(3):236–241 16. Zhang Y, Kutateladze TG (2018) Diet and the epigenome. Nat Commun 9:3375

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17. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293(5532):1074–1080 18. Zhao S, Yue Y, Li YY, Li HT (2019) Identification and characterization of ’readers’ for novel histone modifications. Curr Opin Chem Biol 51:57–65 19. Chan JC, Maze I (2020) Nothing is yet set in (hi)stone: novel post-translational modifications regulating chromatin function. Trends Biochem Sci 45(10):829–844 20. Lang K, Chin JW (2014) Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem Rev 114(9): 4764–4806 21. Xiao H, Schultz PG (2016) At the interface of chemical and biological synthesis: an expanded genetic code. Cold Spring Harb Perspect Biol 8(9):a023945 22. Wang L (2017) Engineering the genetic code in cells and animals: biological considerations and impacts. Accounts Chem Res 50(11): 2767–2775 23. Neumann H, Neumann-Staubitz P, Witte A, Summerer D (2018) Epigenetic chromatin modification by amber suppression technology. Curr Opin Chem Biol 45:1–9 24. No¨dling AR, Spear LA, Williams TL, Luk LYP, Tsai YH (2019) Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells. Essays Biochem 63(2):237–266 25. Chen J, Tsai YH (2022) Applications of genetic code expansion in studying protein posttranslational modification. J Mol Biol 434(8): 167424 26. Neumann H, Peak-Chew SY, Chin JW (2008) Genetically encoding N-epsilon-acetyllysine in recombinant proteins. Nat Chem Biol 4(4): 232–234 27. Neumann H, Hancock SM, Buning R, Routh A, Chapman L, Somers J, OwenHughes T, van Noort J, Rhodes D, Chin JW (2009) A method for genetically installing sitespecific acetylation in recombinant histories defines the effects of H3 K56 acetylation. Mol Cell 36(1):153–163 28. Xie X, Li XM, Qin FF, Lin JW, Zhang G, Zhao JY, Bao XC, Zhu RF, Song HP, Li XD, Chen PR (2017) Genetically encoded photoaffinity histone marks. J Am Chem Soc 139(19): 6522–6525 29. Kim CH, Kang M, Kim HJ, Chatterjee A, Schultz PG (2012) Site-specific incorporation of epsilon-N-crotonyllysine into histones. Angew Chem Int Edit 51(29):7246–7249

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30. Xiao H, Xuan WM, Shao SD, Liu T, Schultz PG (2015) Genetic incorporation of epsilonN-2-hydroxyisobutyryl-lysine into recombinant histones. ACS Chem Biol 10(7): 1599–1603 31. Gattner MJ, Vrabel M, Carell T (2013) Synthesis of epsilon-N-propionyl-, epsilon-Nbutyryl-, and epsilon-N-crotonyl-lysine containing histone H3 using the pyrrolysine system. Chem Commun 49(4):379–381 32. Wilkins BJ, Hahn LE, Heitmuller S, Frauendorf H, Valerius O, Braus GH, Neumann H (2015) Genetically encoding lysine modifications on histone H4. ACS Chem Biol 10(4):939–944 33. Chen XH, Xiang Z, Hu YS, Lacey VK, Cang H, Wang L (2014) Genetically encoding an electrophilic amino acid for protein stapling and covalent binding to native receptors. ACS Chem Biol 9(9):1956–1961 34. Wang ZPA, Kurra Y, Wang X, Zeng Y, Lee YJ, Sharma V, Lin HN, Dai SY, Liu WSR (2017) A versatile approach for site-specific lysine acylation in proteins. Angew Chem Int Edit 56(6): 1643–1647

35. Cigler M, Muller TG, Horn-Ghetko D, von Wrisberg MK, Fottner M, Goody RS, Itzen A, Muller MP, Lang K (2017) Proximitytriggered covalent stabilization of low-affinity protein complexes in vitro and in vivo. Angew Chem Int Edit 56(49):15737–15741 36. Ji Y, Ren C, Miao H, Pang Z, Xiao R, Yang X, Xuan W (2021) Genetically encoding ε-N-benzoyllysine in protein. Chem Commun 57(14): 1798–1801 37. Cao L, Liu J, Ghelichkhani F, Rozovsky S, Wang L (2021) Genetic incorporation of ε-Nbenzoyllysine by engineering methanomethylophilus alvus pyrrolysyl-tRNA synthetase. Chembiochem 22(15):2530–2534 38. Tian HT, Yang JL, Guo AD, Ran Y, Yang YZ, Yang B, Huang RM, Liu HM, Chen XH (2021) Genetically encoded benzoyllysines serve as versatile probes for interrogating histone benzoylation and interactions in living cells. ACS Chem Biol 16(11):2560–2569 39. Willis JCW, Chin JW (2018) Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat Chem 10(8):831–837

Chapter 10 Semisynthesis of Glutamine-Methylated Proteins Enabled by Genetic Code Expansion Weimin Xuan and Xiaochen Yang Abstract Gln methylation is a newly identified histone mark and mediates ribosomal biogenesis. Site-specifically Gln-methylated proteins are valuable tools to elucidate the biological implications of this modification. Herein, we describe a protocol to generate histones with site-specific Gln methylation in a semisynthetic manner. Firstly, an esterified glutamic acid analogue (BnE) is genetically encoded into proteins by genetic code expansion with high efficiency, which can be quantitatively converted into an acyl hydrazide via hydrazinolysis. Then, through a reaction with acetyl acetone, the acyl hydrazide is converted to reactive Knorr pyrazole. Finally, the in situ generated Knorr pyrazole is incubated with methylamine to give Gln methylation. Key words Histone Gln methylation, Genetic code expansion, Glutamic acid analogue, Acyl hydrazide, Knorr pyrazole

1

Introduction To exert or regulate biological functions, proteins in living cells experience extensive posttranslational modifications (PTMs), such as phosphorylation, methylation, acetylation, and glycosylation. The residues that are frequently subject to PTMs include Ser, Thr, Tyr, Lys, and Asn. Particularly due to the advances in mass spectrometry-based proteomic studies, there has been a rapid growth of identified PTM types and sites over the past two decades [1, 2]. Consequently, in-depth investigations are in demand to reveal the detailed mechanisms of these modifications, particularly the ones that are less studied. Notably, PTMs occurring on the side chain of Gln residues have been increasingly identified, including methylation [3], serotonylation [4], and dopaminylation [5]. Among these, Gln methylation was originally discovered on the ribosomal protein of E. coli [6] and later on identified on the GGQ conserved motif of release

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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factor 2 [7, 8]. As a breakthrough, Gln methylation was discovered on histones for the first time by Kouzarides and coworkers as an RNA-polymerase-I dedicated modification and medicates the interactions of facilitator of chromatin transcription (FACT) and nucleosomes [3]. Histone Gln methylation is enriched over the 35S ribosomal DNA transcriptional unit, thereby regulating nucleolar rDNA transcription. Noteworthily in a recent work by Tessarz and coworkers, the ribonucleoprotein Nhp2 was identified as an epigenetic reader of histone Gln methylation (H2A-Q105me) and participates in the coordination of rDNA transcription and the following posttranscriptional processing [9]. Systematic proteomic analysis of protein methylation, along with research on protein Gln methyltransferase, has led to the discovery of more protein substrates subjected to Gln methylation, implying the largely unknown biological functions of this modification [10, 11]. To facilitate the fundamental studies of Gln methylation, methods that allow the site-specific generation of methylated Gln proteins are demanded. To this end, Liu group chemically synthesized histone protein with site-specific Gln methylation [12], and Davis group developed a C(sp3)-C(sp3) bond-forming reaction to derivatize an elegantly introduced dehydroalanine residue for this purpose [13]. Recently, we reported an innovative strategy for sitespecific and efficient generation of Gln methylation on proteins [14]. It involves the genetic incorporation of L-glutamic acid γ-benzyl ester (BnE) with an evolved orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair and the subsequent conversion of BnE to methylated Gln via acyl hydrazide and Knorr pyrazole intermediates. Here, we provide a stepwise protocol (Fig. 1) for the generation of Gln methylation on a model protein (i.e., H3-Q56me), hoping to aid the further investigation of this PTM.

2

Materials Commercially available chemicals and reagents are used without pretreatment.

2.1 Recombinant Expression of BnEContaining Proteins

1. E. coli BL21(DE3). 2. Two plasmids, i.e., pUltra-BnERS (SpecR) and pET22b-T5H3-Q56TAG (AmpR), are used to express H3-Q56BnE (see Note 1). BnERS has three mutation sites (N311S, C313A, and Y349F) compared to the wide-type MbPylRS. The sequences of BnERS and tRNAPyl are shown in Fig. 2. 3. L-Glutamic acid γ-benzyl ester (BnE) is commercially available. 4. 10% (v/v) glycerol: 100 mL glycerol in 900 mL ddH2O. Sterilize by autoclaving for 15 min at 121 °C and 15 psi. Store at 4 °C.

Site-Specific Gln by Genetic Code Expansion

Fig. 1 Workflow of semisynthesis of H3-Q56me

Fig. 2 The sketch maps of the used two plasmids and the sequences of BnERS and tRNAPyl

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5. Super-optimal broth (SOB) medium: 20 g/L tryptone, 5 g/L yeast extract, 8.5 mM NaCl, 2.5 mM KCl, 10 mM MgCl2. Dissolve 4 g tryptone, 1 g yeast extract, 0.1 g NaCl, 0.037 g KCl, and 0.19 g MgCl2 in 200 mL ddH2O. Sterilize by autoclaving for 15 min at 121 °C and 15 psi. Store at room temperature. 6. Lysogeny broth (LB) agar: 10 g/L tryptone, 5 g/L yeast extract,10 g/L NaCl, and 15 g/L agar. Dissolve 5 g tryptone, 2.5 g yeast extract, 5 g NaCl, and 7.5 g agar in 500 mL ddH2O. Sterilize by autoclaving for 15 min at 121 °C and 15 psi. Store at room temperature. 7. Lysogeny broth (LB) medium: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. Dissolve 5 g tryptone, 2.5 g yeast extract, and 5 g NaCl in 500 mL ddH2O. Sterilize by autoclaving for 15 min at 121 °C and 15 psi. Store at room temperature. 8. 2-YT broth medium: 16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl. Dissolve 8 g tryptone, 5 g yeast extract, and 2.5 g NaCl in 500 mL ddH2O. Sterilize by autoclaving for 15 min at 121 °C and 15 psi. Store at room temperature. 9. 1 M IPTG stock (1000×): Dissolve 2.38 g isopropyl β-Dthiogalactopyranoside (IPTG) in ddH2O to a final volume of 10 mL. Sterilize through a 0.22 μm syringe filter. Store at 20 °C. 10. Antibiotic stocks: 100 mg/mL ampicillin (Amp, 1000×); 50 mg/mL spectinomycin (Spec, 1000×). Dissolve 1 g Amp or 0.5 g Spec in ddH2O to a final volume of 10 mL. Sterilize through a 0.22 μm syringe filter. Store at -20 °C. 2.2 Protein Purification from Expression Host

1. Phosphate-buffered saline (PBS): 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4. Store at room temperature. 2. Lysis buffer: 100 mM Na2HPO4, 6 M Gn·HCl, pH 8.0. Store at 4 °C. 3. Washing buffer: 100 mM Na2HPO4, 10 mM imidazole, 6 M Gn·HCl, pH 8.0. Store at 4 °C. 4. Elution buffer: 100 mM Na2HPO4, 250 mM imidazole, 6 M Gn·HCl, pH 8.0. Store at 4 °C. 5. H3 stock buffer: PBS with 6 M Gn·HCl, pH 7.4. Store at 4 °C. 6. Concentration tube, 4 mL, 10-kDa cutoff.

2.3 Reactions Performed on H3Q56BnE

1. Hydrazine: 80% (v/v) NH2NH2·H2O 80%. 2. Acetyl acetone (acac). 3. 2 M methylamine: Dissolve 52 mg methylamine hydrochloride in 400 μL DMSO.

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4. Acidic denaturing phosphate solution: 20 mM Na2HPO4, 4 M Gn·HCl, pH 3. 5. Phosphate buffer: 200 mM Na2HPO4, pH 8. 6. Denaturing phosphate buffer: 20 mM Na2HPO4, 6 M Gn·HCl, pH 8. 7. Dialysis membrane tube, 3500 MWCO, φ22, MD: 34 mm. 8. Concentration tube, 4 mL, 3-kDa cutoff.

3

Methods During electrocompetent cell preparation, plasmid transformation, and protein expression, handle bacteria cultures under sterile conditions at all times.

3.1 Preparation of Electrocompetent Cells

Ten percent glycerol, centrifuge bottles, pipette tips, and 0.6 mL centrifuge tubes are sterilized and prechilled. 1. Pick a single colony of E. coli BL21(DE3). Inoculate into 4 mL of 2-YT and grow at 37 °C and 220 rpm overnight. 2. Inoculate 1 mL of fresh E. coli BL21(DE3) culture to 1 L of sterilized SOB culture in a 5-L flask. 3. Grow the cells in a shaker at 37 °C 220 rpm to an OD600 of approximately 0.5–0.7 (see Note 2). 4. Immediately chill the cells on ice for about 20 min. Swirl the bottles every 3–4 min. For all subsequent steps, keep the cells on ice as much as possible. 5. Spin the cells in centrifuge bottles at 4 °C and 6400 g for 7 min. Carefully decant the supernatant. 6. Resuspend the cell pellets with totally 1 L of 10% glycerol by rigorous shaking with hands (see Note 3). Centrifuge at 4 °C and 6400 g for 7 min. Carefully decant the supernatant. 7. Repeat step 6. 8. Resuspend the cells with 450 mL of 10% glycerol by rigorous shaking with hands. Centrifuge at 4 °C and 6400 g for 7 min. Carefully decant the supernatant. 9. Resuspend the cell pellet with 1–2 mL of 10% glycerol by gently washing the pellet in a repeated manner using a pipette (see Note 4). 10. Aliquot the cell solution 50 μL per sample into 0.6 mL centrifuge tubes. Immediately freeze the cells with liquid nitrogen. Store at -80 °C.

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3.2 Plasmid Transformation

1. Take one tube of electrocompetent BL21(DE3) cells from 80 °C refrigerator, and immediately put it on ice to thaw. Prechill a 0.1 cm electroporation cuvette on ice (see Note 5). 2. Add 100 ng of each plasmid (i.e., pUltra-BnERS and pET22bT5-H3-Q56TAG) to the thawed competent cells, and gently mix by pipetting (see Note 6). 3. Transfer the cell plasmid mixture to the chilled electroporation cuvette and gently tap the suspension to the bottom. Place the cuvette into MicroPulser electroporator set at 1.8 kV, pulse once (see Note 7). 4. After pulse, immediately add 1 mL of SOB medium to the cuvette. Quickly but gently mix the cells with a pipette. Transfer the cells to a 12 mL culture tube and then recover in a shaker at 37 °C and 220 rpm for 1 h (see Note 8). 5. Spread 50–100 μL of cells on an LB agar plate containing Amp and Spec, and incubate at 37 °C for 12 h (see Note 9).

3.3 Protein Expression

1. Pick a single colony and inoculate into 3 mL of LB broth (Amp and Spec) in a 12 mL culture tube. Incubate in a shaker at 37 ° C and 220 rpm for 10 h. 2. Take 400 μL of the resulting culture and dilute to 40 mL (100-fold) with 2-YT broth medium (Amp and Spec). Grow cells in a shaker at 37 °C and 220 rpm until the OD600 reaches 0.7. 3. At this optical density, add 76 mg of BnE powder into the culture to the final concentration of 8 mM (see Note 10). Grow the culture for another 15 min before induction with IPTG (final concentration = 1 mM). 4. The expression last 10 h in a shaker at 37 °C and 220 rpm.

3.4 Protein Purification

The construct of H3-Q56TAG contains a C-terminal 6xhistidine tag, allowing affinity purification with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin. 1. Centrifuge the cells at 4 °C and 14,400 g for 15 min in a 50 mL centrifuge tube. 2. Carefully decant the supernatant. Resuspend the cells with 10 mL of PBS by vortexing. 3. Lyse the cells in a 50 mL centrifuge tube with a sonic membrane disruptor using 50% power (2 s on/2 s off, 20 min). During lysis, the 50 mL centrifuge tube was held in an ice bath. If the resulting cell lysate is viscous, repeat the sonication until a homogeneous solution is achieved. 4. Centrifuge the cell lysate at 4 °C and 14,400 g for 30 min. Carefully decant the supernatant (see Note 11).

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5. Resuspend the pellet with 30 mL of lysis buffer. Put the tube in a rotary mixer for 40 min to completely dissolve the pellet at room temperature (see Note 12). 6. Repeat the above centrifugation step (see Note 13). Carefully transfer the supernatant to a new 50 mL centrifuge tube. 7. Add 200 μL of Ni-NTA resin slurry to the supernatant and keep the mixture on a rotary mixer for 30 min at room temperature. 8. Load the resin mixture on a 6 mL gravity column and drain out the culture. 9. Wash the resin with 120 mL of washing buffer. 10. Elute protein from the resin with 1 mL of elution buffer (see Note 14). 11. Buffer-exchange the collected protein solution with 12 mL of H3 stock buffer using a 4 mL 10-kDa cutoff concentration tube. At this time point the quality of purified proteins should be analyzed by SDS-PAGE and mass spectrometry, and the protein concentration should be measured with by BCA assay according to the manufacture’s protocol (see Note 15). 3.5 Conversion of BnE on a Protein to Methylated Gln

1. Add 50 μL of hydrazine (80% v/v) to 150 μL of 12 μM H3-Q56BnE in H3 stock buffer. The final concentration of NH2NH2·H2O is 20% (v/v). Incubate the reaction mixture at 37 °C for 1.5 h. 2. Dialyze the resulting reaction mixture with 1 L acidic denaturing phosphate solution for four times (250 mL × 4, 2 h for each) at room temperature to remove the excessive hydrazine (see Note 16). At this time point the generation of acyl hydrazide can be characterized by mass spectrometry. 3. Concentrate the protein solution with a 4 mL 3-kDa cutoff concentration tube to approximately 200 μL. 4. To the above protein solution is added 4 μL of acac to a final concentration of 200 mM. Incubate the reaction mixture at 37 °C for 1.5 h. The generation of Knorr pyrazole intermediate can be characterized by mass spectrometry (see Note 17). 5. Add 200 μL of denaturing phosphate buffer to the above reaction mixture to adjust the pH, and add 400 μL of methylamine stock solution to the reaction mixture at a final concentration of 1 M. Adjust the pH to 9 with 5 M NaOH(aq). Incubate the reaction mixture at 37 °C for 5 h to afford the final product (see Note 18). The formation of H3-Q56me can be characterized by mass spectrometry.

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Notes 1. In pair with tRNAPyl, an evolved Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS) variant (i.e., BnERS, harbored in pUltra) is used to encode BnE [15]. The H3 gene construct with an amber codon at Q56 (H3-Q56TAG) is cloned in vector pET22b-T5. 2. It is best to harvest the cells at early- to mid-log phase. 3. If it takes a long time to resuspend the cells, put the centrifuge bottles on ice every 1–2 min to avoid warm-up of the cells. 4. Do not spear the pellet. Be patient. Finally, you should get a homogeneous cell solution. 5. The competent cells must thaw on ice to ensure their vitality. 6. For electroporation, plasmids should be stored in a low ionic strength buffer such as DNase/RNase-free water. 7. Place the cuvette in the chamber slide with care and push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. 8. The period between applying the pulse and transferring the cells to the outgrowth medium is crucial for recovering E. coli transformants. Delaying this transfer by even 1 min causes a threefold drop in transformation efficiency. This decline continues to a 20-fold drop by 10 min. 9. Electroporation usually shows high efficiency, even for simultaneous transformation of two plasmids; 50–100 μL of cell cultures is enough to provide the number of colonies. Further, 12-h incubation is enough to get the desired colonies. 10. Using BnE powder is critical for efficient incorporation, as the ester bond is susceptible to hydrolysis in aqueous solution. Using BnE stock solution under basic conditions can completely fail the expression. 11. Histones do not fold properly in bacteria and exist in the form of inclusion bodies. 12. To avoid the adverse effect of urea to the following reactions, Gn·HCl was chosen as denaturing agent instead of urea. 13. All the above operations are carried out in a single 50 mL centrifuge tube. 14. Elution buffer should be gently applied to minimize resin disruption. 15. Since the protein is detected by mass spectrometry under denaturing conditions, there is no dimerization. 16. To remove excessive hydrazine, dialysis should be used instead of a concentration tube.

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17. The Knorr pyrazole intermediate is not stable, so use it immediately after preparation. 18. During the reaction, the pH of the system may decrease; to make sure the pH of reaction mixture is at approximately 9, carefully check the solution pH by applying 1 μL of reaction mixture to precision pH test strips.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21807061), Natural Science Foundation of Tianjin (18JCYBJC41600), Nankai University, and Tianjin University. References 1. Zhao YM, Garcia BA (2015) Comprehensive catalog of currently documented histone modifications. Cold Spring Harb Perspect Biol 7(9):a025064. https://doi.org/10.1101/ cshperspect.a025064 2. Andrews FH, Strahl BD, Kutateladze TG (2016) Insights into newly discovered marks and readers of epigenetic information. Nat Chem Biol 12(9):662–668. https://doi.org/ 10.1038/nchembio.2149 3. Tessarz P, Santos-Rosa H, Robson S, Sylvestersen KB, Nelson CJ, Nielsen ML, Kouzarides T (2014) Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification. Nature 505(7484):564–568. https:// doi.org/10.1038/nature12819 4. Farrelly LA, Thompson RE, Zhao S, Lepack AE, Lyu Y, Bhanu NV, Zhang B, Loh Y-HE, Ramakrishnan A, Vadodaria KC, Heard KJ, Erikson G, Nakadai T, Bastle RM, Lukasak BJ, Zebroski H III, Alenina N, Bader M, Berton O, Roeder RG, Molina H, Gage FH, Shen L, Garcia BA, Li H, Muir TW, Maze I (2019) Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature 567(7749):535–539. https://doi.org/10.1038/s41586-0191024-7 5. Lepack AE, Werner CT, Stewart AF, Fulton SL, Zhong P, Farrelly LA, Smith ACW, Ramakrishnan A, Lyu Y, Bastle RM, Martin JA, Mitra S, O’Connor RM, Wang Z-J, Molina H, Turecki G, Shen L, Yan Z, Calipari ES, Dietz DM, Kenny PJ, Maze I (2020) Dopaminylation of histone H3 in ventral tegmental area regulates cocaine seeking. Science 368(6487):197–201. https://doi.org/10. 1126/science.aaw8806

6. Lhoest J, Colson C (1977) Genetics of ribosomal protein methylation in Escherichia coli. Mol Gen Genet 154(2):175–180. https://doi. org/10.1007/BF00330833 7. Dinc¸bas-Renqvist V, Engstro¨m Å, Mora L, Heurgue´-Hamard V, Buckingham R, Ehrenberg M (2000) A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation. EMBO J 19(24):6900–6907. https://doi.org/10.1093/emboj/19.24. 6900 8. Nakahigashi K, Kubo N, Narita S-i, Shimaoka T, Goto S, Oshima T, Mori H, Maeda M, Wada C, Inokuchi H (2002) HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination. Proc Natl Acad Sci 99(3): 1473–1478. https://doi.org/10.1073/pnas. 032488499 9. Mawer JSP, Massen J, Reichert C, Grabenhorst N, Mylonas C, Tessarz P (2021) Nhp2 is a reader of H2AQ105me and part of a network integrating metabolism with rRNA synthesis. EMBO Rep 22(10):e52435. https://doi.org/10.15252/embr.202152435 10. Kusevic D, Kudithipudi S, Jeltsch A (2016) Substrate specificity of the HEMK2 protein glutamine methyltransferase and identification of novel substrates. J Biol Chem 291(12): 6124–6133. https://doi.org/10.1074/jbc. M115.711952 11. Zhang M, Xu J-Y, Hu H, Ye B-C, Tan M (2018) Systematic proteomic analysis of protein methylation in prokaryotes and eukaryotes revealed distinct substrate specificity.

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Proteomics 18(1):1700300. https://doi.org/ 10.1002/pmic.201700300 12. He Q, Li J, Qi Y, Wang Z, Huang Y, Liu L (2017) Chemical synthesis of histone H2A with methylation at Gln104. Sci China Chem 60(5):621–627. https://doi.org/10.1007/ s11426-016-0386-4 13. Wright TH, Bower BJ, Chalker JM, Bernardes GJL, Wiewiora R, Ng WL, Raj R, Faulkner S, Vallee MRJ, Phanumartwiwath A, Coleman OD, Thezenas ML, Khan M, Galan SRG, Lercher L, Schombs MW, Gerstberger S, Palm-Espling ME, Baldwin AJ, Kessler BM, Claridge TDW, Mohammed S, Davis BG (2016) Posttranslational mutagenesis: a

chemical strategy for exploring protein sidechain diversity. Science 354(6312):11. https://doi.org/10.1126/science.aag1465 14. Yang X, Miao H, Xiao R, Wang L, Zhao Y, Wu Q, Ji Y, Du J, Qin H, Xuan W (2021) Diverse protein manipulations with genetically encoded glutamic acid benzyl ester. Chem Sci 12(28):9778–9785. https://doi.org/10. 1039/d1sc01882e 15. Chatterjee A, Sun SB, Furman JL, Xiao H, Schultz PG (2013) A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry 52(10):1828–1837. https://doi.org/10. 1021/bi4000244

Part II Applications in Mammalian Cells

Chapter 11 Genetic Code Expansion in Mammalian Cells Zhigang Wu and Jie Wang Abstract The expansion of the genetic code has enabled the incorporation of noncanonical amino acids (ncAAs) into a defined site of proteins. By introducing such a unique handle into the protein of interest (POI), bioorthogonal reactions can be utilized in live cells to monitor or manipulate the interaction, translocation, function, and modification of the POI. Here, we describe a basic protocol outlining the necessary steps to incorporate a ncAA into a POI in mammalian cells. Key words Genetic code expansion, Non-canonical amino acids, Mammalian cells, Cell transfection, Stable cell line

1

Introduction Genetic code expansion is a valuable tool to study and manipulate protein function in living cells. During the past decades, genetically encoded noncanonical amino acids (ncAAs) have been widely used to study the spatiotemporal resolution of protein function, proteinprotein interaction, protein dynamic posttranslational modifications, protein translocation, and signal transduction in mammalian cells [1]. Genetic code expansion also opens new avenues for clinical therapeutics of protein-mediated diseases [2]. For example, Shi et al. reported that ncAAs can be incorporated in nonsense mutation in the dystrophin gene to restore the full-length protein and muscle function [3]. The Liu group reported ncAA-triggered therapeutic switch can fast express protein to alleviate hyperglycemia in diabetic mice [4]. These results demonstrate potential therapeutic uses of ncAAs. Importantly, incorporation and expression of ncAAs in mammalian cells is the crucial basis. Site-specific incorporation of a ncAA in mammalian cells is achieved by an orthogonal aaRS/tRNA pair in response to a blank codon [1]. In 2002, Yokoyama et al. firstly showed that Escherichia coli tyrosyl-synthetase (TyrRS) and Geobacillus

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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stearothermophilus tRNA can be utilized to incorporate 3-iodo-Ltyrosine into proteins in CHO and HEK293T cells [5]. In the next two decades, TyrRS from Methanococcus jannaschii (Mj), PylRS from Methanosarcina barkeri (Mb) and Methanosarcina mazei (Mm), LeuRS from E. coli, PylRS-based chimeric aaRS/tRNA pairs, etc. have been successfully developed for ncAA incorporation in mammalian cells [1]. Besides, the codon for ncAAs has also been expanded from the amber stop codon to ochre/opal stop codon, quadruplet codons, and the reassigned “sense” codons [1]. As the PylRS/tRNA pair are naturally utilized to encode pyrrolysine in response to the amber codon, it is orthogonal to other aaRS/tRNA pairs in most species [1]. Thus, the PylRS/tRNA pair can be evolved in E. coli and further used in other species, which boosts the exploration of new ncAAs and their applications. Here, we describe a detailed protocol for incorporation of a ncAA by PylRS/tRNA pair in response to the amber codon into a protein of interest (POI) in mammalian cells (see Note 1).

2

Materials

2.1 Materials for Molecular Biology

1. P10/P20/P200/P1000 pipette tips. 2. 1.5 mL microcentrifuge tubes. 3. 15 and 50mL centrifuge tubes. 4. Agarose for DNA gels. 5. 10,000  nucleic acid stain (e.g., Ultra GelRed). 6. DNA gel extraction kit. 7. Plasmid extraction kit, ideally endotoxin-free. 8. Site-directed mutagenesis kit.

2.2 Materials for Cell Culture

1. HEK293T cells. 2. Fetal bovine serum (FBS). 3. DMEM containing 10% (v/v) FBS: Add 45 mL of FBS to 405 mL of DMEM, and mix well. This medium can be stored at 4  C for 6 months. 4. Trypsin (e.g., Gibco #25200-072). 5. Penicillin-streptomycin (e.g., Gibco #15140-122). 6. Poly-D-lysine (CAS: 27964-99-4; e.g., Sigma, #P6407). 7. 6-, 12-, and 24-well cell culture plates. 8. 100  20 mm cell culture dishes. 9. 10, 25, and 50 mL serological pipets. 10. Phosphate-buffered saline (PBS).

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11. Protein 5  loading buffer: Mix 2.5 mL of Tris–HCl (pH 6.8), 1 g of SDS, 50 mg of bromophenol blue, 500 μL of β-mercaptoethanol, and 5 mL of glycerol in a 15 mL tube. Then, add water to a final volume to 10 mL. Mix the solution well and divide the buffer into 1 mL aliquots. These buffer can be stored at 20  C for several months. 12. 100 mM BocK: Dissolve 246 mg of N-ε-Boc-L-lysine (CAS: 2418-95-3) in 10 mL of 100 mM HCl to make a 100 mM stock. This can be divided into 1 mL aliquots and stored at 20  C for 6 months. 13. FAST transfer buffer stock solution (10) (e.g., Biofuraw #8006-6006). 14. Simple PAGE Bis-Tris (e.g., Sangon Biotech #C6912020001). 15. TBST (10) (e.g., Sangon Biotech #C006161). 16. Mammalian protein extraction buffer (e.g., Thermo Fisher #78501). 17. Cell counting kit 8 (CCK8). 18. Microplate reader. 2.3 Materials for Transfection

1. OPTI-MEM. 2. Transfection reagent (e.g., Lipofectamine 3000). 3. 0.45 μm sterile syringe filters. 4. Polybrene. 5. Puromycin dihydrochloride. 6. Plasmid pCMV-MbPylRS (Addgene, #91706). 7. Plasmid pLX304 (Addgene, #25890). 8. Plasmid pCMV-dR8.2 dvpr (Addgene, #8455). 9. Plasmid pVSV-G (Addgene, #36399).

3

Methods

3.1 Preliminary Test for Incorporation of a ncAA into the POI in Mammalian Cells 3.1.1 Demonstrate the Solubility and Cytotoxicity of ncAAs

1. Here we use N-ε-Boc-L-lysine (BocK) as an examplar ncAA. Test the solubility of BocK by diluting the 100 mM stock solution to 0.1, 1, and 10 mM with PBS (see Note 2). 2. Plate 4  104 HEK293T cells in a 96-well plate, and then incubate cells at 37  C in 5% CO2 overnight. Afterwards, add a different amount of BocK stock solution into cell culture medium with the final concentration of BocK at 100 μM, 500 μM, 1 mM, 2 mM, and 5 mM. After 48-h incubation, add CCK8 reagent and measure the absorbance at 460 nm to demonstrate the cytotoxicity of BocK.

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3.1.2 Demonstrate the Incorporation of BocK on a Model Protein (See Note 3)

1. Here we use sfGFP-149TAG as an example of model proteins. Firstly, synthesize or clone the sfGFP-149TAG gene and insert the gene into a eukaryotic vector for mammalian cell (such as pCMV or pCDNA 4.1), and add a His tag or Flag tag sequence in the C-terminal of model protein (see Note 4). 2. Prepare the plasmid encoding the PylRS and tRNA (see Note 5). In this section, we take the BocK as an example, and the BocK can be recognized by the WT-MbPylRS. The plasmid encodes WT-MbPylRS can be generated from the pCMVMbPylRS by site-directed mutagenesis. 3. Add 1 mL of 0.1 mg/mL poly-D-lysine solution to one well of a 6-well plate for 10 min and then remove the solution. Afterward, plate 1.2  106 HEK293T cells in one well of the well, and then incubate cells at 37  C in 5% CO2 overnight. Prepare three wells with HEK293T cells as described and check the confluency in the next day to make sure the confluency is 70–80%. 4. Remove the culture medium and then add 2 mL fresh culture medium and 20 μL of 100 mM BocK stock solution. Subsequently, incubate the cells at 37  C with 5% CO2 during the preparation of transfection mixtures (see Note 6). Prepare one well with BocK treatment and two wells without BocK. 5. Prepare the transfection mixtures. For each well, mix 4 μL of Lipofectamine 3000 and 125 μL of Opti-MEM culture medium to get reagent 1 and mix 4 μL of P3000, 125 μL of Opti-MEM, and 2 μg of plasmid encoding PylRS/tRNA pair and 2 μg of plasmid encoding POI to get reagent 2. After 5 min, mix reagent 1 and 2 by pipetting up and down slightly to get the transfection mixtures. After incubating the transfection mixtures for 15 min at room temperature, the mixture is added to a 6-well plate for further culture (Fig. 1). Prepare transfection mixtures for two wells: one well is used for the BocK treatment group (positive group) and the other one is used for the Bock-free group (for checking the orthogonality of PylRS mutant to the endogenous aaRS). The third well without BocK treatment and any transfection is used as a blank control. 6. Incubate the transfected cells with or without BocK treatment as well as the blank control cells for 24–48 h at 37  C with 5% CO2, then harvest the cells, and analyze the protein of interest by immunoblotting (see Note 4).

3.1.3 For SDS Page and Immunoblotting Analysis

1. Remove DMEM culture medium and wash cells with 1 mL of cold PBS. Next, add 200 μL of mammalian protein extraction buffer (see Note 7) directly to each well, put the plate on ice, and rock for 20 min. Subsequently, collect the cell lysate and add 50 μL of 5  loading buffer. Heat at 95  C for 10 min.

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Fig. 1 Plasmid map and general scheme for ncAA incorporation in mammalian cells. (a) pCMV plasmid encoding the POI containing TAG and pCMV plasmid encoding PylRS and 4  tRNA. (b) The procedure of incorporating a ncAA into the POI in mammalian cells by transfection. (c) Analysis of the POI by western blot

2. Load 10 μL protein solution from each well into two lanes of a 10-well SDS-PAGE gel. After electrophoresis, transfer proteins from the gel to a PVDF membrane for further immunoblotting analysis. For each protein studied, three groups should be conducted in parallel: the “+ncAA” experimental group, “ ncAA” control group, and the “blank” control group (see Note 8). 3.2 Genetic Code Expansion in Mammalian Cells by the Two-Plasmid System

1. To prepare the plasmid that encodes the POI with an amber codon, firstly clone the gene into a vector containing a C-terminal tag. For example, we use pCMV-C-His to generate the plasmid pCMV-POI-His. The gene should terminate with either TAA or TGA stop codon.

3.2.1 Incorporation of a ncAA in Mammalian Cells

2. The other protocol of incorporation and expression of ncAA by transient transfection refers to Subheading 3.1 and the experimental workflow as outlined in Fig. 1.

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3.3 Genetic Code Expansion in Mammalian Cells via Stable Cell Line

For the frequently used PylRS/tRNA pair, we can establish a cell line that can stably express the aaRS and tRNA. By this way, we only need to transfect plasmid encode POI with amber codon for further study. The stable aaRS/tRNA expressed in the cell line can be constructed as follows.

3.3.1 Preparation of the Lentivirus

1. Culture the HKE293T cell in a 12-well plate and until the confluence reaches 60–70%, and change the culture medium to the fresh culture medium without penicillin-streptomycin and fetal bovine serum. 2. The 3 μL of P3000 reagent, 100 μL of Opti-MEM, and 3 μL of Lipofectamine 3000 reagent are mixed first, and then a mixture of the plasmids pLX304-EGFP-P2A-PylRS (0.5 μg), pCMV-dR8.2 (0.5 μg), and pVSV-G (0.4 μg) into Opti-MEM mix is added to form the transfection mixture. Afterwards, the transfection mixture is incubated for 15 min and then added to the 12-well plate for transfection. 3. After culture for 48 h, the culture medium, which contains the lentivirus, is collected and filtered through a 0.45 μm filter to obtain the lentivirus solution for the stable cell line construction (Fig. 2). If the titer of lentivirus is not high enough, we can concentrate the lentivirus by centrifugation.

3.3.2 Preparation of the Stable Cell Line

1. Add 0.4 mL of the collected medium (containing lentivirus) into fresh HEK293T cells at 70–80% confluence in three 6 cm dishes for 48-h incubation at 37  C in 5% CO2. 2. Next, replace the culture medium with fresh medium containing 1 μg/mL, 3 μg/mL, and 10 μg/mL puromycin every day for selection. 3. After a week, cells expressing PylRS with EGFP can be sorted by flow cytometry. 4. Plate about 1  102 sorted cells to a 10 cm dish and culture them for 3 days; afterwards, 3–5 different monoclonal cell cluster are picked by tips for further culture. 5. All the picked monoclonal cell line should be verified to check if the PylRS and tRNA are stably expressed.

4

Notes 1. The wild-type PylRS/tRNA pairs can incorporate a number of pyrrolysine analogues, and the evolved PylRS/tRNA pairs can also incorporate Phe and Tyr analogues. In addition, ncAAs

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Fig. 2 The general scheme for genetic code expansion in a stable cell line

with bulky groups at the Lys side chains (e.g., cyclooctyne or azidobenzyloxycarbonyl) can be incorporated into a POI by PylRS/tRNA variants. Overall, the PylRS/tRNA pairs have a wide substrate scope of ncAAs. Here, we use the wild-type PylRS and one of its substrates (i.e., BocK) as the example to describe a detailed protocol for incorporation of a ncAA into a POI in mammalian cells. 2. Some of the ncAAs with poor solubility could be dissolved in 200 mM NaOH or 200 mM HCl to obtain the 100 mM stock solution. If the ncAA is dissolved in ddH2O, filtration through a 0.22 μm filter is needed. Generally, the working concentration of ncAAs ranges from 0.1 to 2 mM. 3. Incorporation efficiency of a ncAA is dependent on the incorporation site of the POI. Thus, we recommend performing some preliminary tests to validate each required component, even though the aaRS variant or ncAA is reported in the literatures.

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4. Addition of a fused tag (e.g., His tag, Flag tag) at the protein C-terminal enables detection of the full-length protein, whereas a N-terminal tag can report both the truncated fragment and the full-length product. 5. Usually, the plasmid maps are available in literatures or Addgene (e.g., https://www.addgene.org/collections/ genetic-code-expansion/). Plasmid pCMV-MbPylRS is available from Addgene (#91706). We just need to mutate the PylRS gene to our desired one to recognize the corresponding ncAA. The mutations of PylRS can be conducted by sitedirected mutagenesis. 6. If the ncAA stock solution is in NaOH or HCl, it may be necessary to adjust the pH after adding the ncAA stock solution to the culture media. 7. RIPA lysis solution is often used for mammalian protein extraction. It contains 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. Commercially available lysis buffer (e.g., Thermo Fisher #78501) for mammalian protein extraction should also work for most experiments. 8. The user may suffer from the problem of low expression level of POI containing the ncAA. We can try these methods from the following reference to improve the efficiency of ncAA incorporation. (1) multiple copies of tRNA (such as, 6–12 copies of tRNA) [4, 6], (2) increasing the concentration of ncAAs, (3) adding a nuclear localization sequence on PylRS, and (4) using the chimeric PylRS of Mm and Mb [7].

Acknowledgements This work was supported by the National Natural Science Foundation of China (22077059, 22277048), Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20210324104210028, RCYX20210609103118010). References 1. Dumas A, Lercher L, Spicer CD et al (2015) Designing logical codon reassignmentexpanding the chemistry in biology. Chem Sci 6:50–69 2. Chin JW (2017) Expanding and reprogramming the genetic code. Nature 550:53–60

3. Shi N, Yang Q, Zhang H et al (2022) Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids. Nat Biomed Eng 6:195–206

Genetic Code Expansion in Mammalian Cells 4. Chen C, Yu G, Huang Y et al (2022) Geneticcode-expanded cell-based therapy for treating diabetes in mice. Nat Chem Biol 18:47–55 5. Sakamoto K, Hayashi A, Sakamoto A et al (2002) Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. Nucleic Acids Res 30:4692–4699

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6. Ryu Y, Schultz PG (2006) Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat Methods 3:263–265 7. Bryson DI, Fan C, Guo LT et al (2017) Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol 13:1253–1260

Chapter 12 Generation of Amber Suppression Cell Lines Using CRISPR-Cas9 Birthe Meineke and Simon J. Els€asser Abstract Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid. Key words Genetic code expansion, Amber suppression, Mammalian cell culture, Non-canonical amino acids, Unnatural amino acids, CRISPR-Cas9, tRNA, Genome editing

1

Introduction Genetic code expansion via amber suppression is a powerful tool to introduce site-specific, genetically encoded nonnatural chemical groups into proteins of interest. A widely used amber suppression system exploits the pyrrolysine-tRNA (PylT) and its cognate aminoacyl-tRNA-synthetase (PylRS) from methanogenic archaea for incorporating a noncanonical amino acid (ncAA) in response to a premature amber stop codon (TAG) in the coding sequence of a protein of interest (POI). We have previously described a versatile two-plasmid system to co-express the Methanosarcina mazei (Mma) PylT/RS pair and a gene of interest (GOI) [1–4]. High gene dosage and efficient amber suppression is achieved by transient transfection. Genomic integration of PylT/RS and PylT/

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_12, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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GOI plasmids using piggyBac transgenesis allows for amber suppression in stable cell lines [1, 5]. Generally, genomic integration yields lower expression levels compared to transient transfection. However, PylRS expression after piggyBac integration supported suppression efficiency comparable to transient transfection [3]. PylRS expressed from the ROSA26 locus in mouse embryonic stem cells was sufficient to sustain amber suppression when tRNA and GOI were transiently transfected or integrated by piggyBac transgenesis [6]. Hence, generation of a PylRS-expressing cell line, into which a PylT/GOI plasmid is transiently transfected provides a useful strategy to increase transfection efficiency of the GOI and more comparable and reproducible results [3]. PiggyBac-mediated transposition is an untargeted process that yields one or more integrations at random genomic loci; the exact copy number and place in the genome are not controlled. Here we provide an alternative protocol which enables one-step integration of the PylT/PylRS expression cassette from our plasmid system into a defined genomic locus. We selected AAVS1 (adenoassociated integration site 1) locus on chromosome 19, which has been used successfully as an integration site for donor DNA by CRISPR-Cas9 targeting [7]. AAVS1 is considered a “safe harbor” in the human genome, i.e., a genomic locus that can be disrupted without negatively affecting the cell, while allowing expression of integrated genes [8]. Modern CRISPR-Cas9-based approaches build on versatile tools for genomic manipulation (reviewed in e.g., [9, 10]). A convenient and adaptable protocol, based on the type II CRISPR system from Streptococcus pyogenes, has been described by Feng Zhang and colleagues [10]. S. pyogenes CRISPR-Cas9 for use in mammalian cells consists of 20-nucleotide-long CRISPR RNAs (crRNAs), an auxiliary transactivator RNA (tracrRNA), and the Cas9 protein. Both RNA components can be fused to generate a single chimeric guide RNA (sgRNA) when used in mammalian genome editing. The RNA components guide the Cas9 nuclease to DNA, to induce double-strand breaks (DSBs). The sequences targeted by S. pyogenes crRNA are 20 nucleotides long, match the crRNA sequence, and are invariantly followed by a 5′NGG PAM (protospacer adjacent repeat). Cas9 nuclease cleavage occurs in the targeted sequence, three nucleotides 5′ of the PAM. For expression in mammalian cells, plasmids were developed that combine U6 promoter-driven sgRNA expression and CMV promoter-regulated expression of human codon-optimized S. pyogenes Cas9 [10]. Upon expression in the target cell, sgRNA/Cas9 induces sitespecific genomic DSBs. Gene editing can then occur during the process of cellular DSB repair, most often through either one of the major DSB repair pathways: homology-driven repair (HDR) or nonhomologous end joining (NHEJ). The NHEJ pathway is

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typically the predominant repair mechanism, and it can be utilized to insert exogenous linear DNA fragments into the DSB site. The NHEJ-mediated repair pathway may lead to a processing of the broken DNA before ligation, leading to insertions or deletions (indels) at the repaired sites. Despite this, large DNA fragments (up to 32 kb) can be reliably inserted at CRISPR-Cas9-induced genomic DSBs using NHEJ-based approaches [11]. In practice, NHEJ-mediated knock-in using CRISPR-Cas9 targeting can be achieved by cotransfecting three plasmids: one sgRNA/Cas9 plasmid targeting a genomic locus and a second sgRNA/Cas9 plasmid to introduce one or more DSB in the third, donor, plasmid. In their study, He et al. explore two approaches for donor plasmid linearization: either the entire plasmid is integrated after single-incision linearization or a dualtargeting sgRNA, resulting in double cleavage of the donor plasmid, generating two plasmid fragments of which either one may be integrated [11]. In this protocol, we adapted this approach to knock in either entire linearized pAS or the PylRS expression/ selection cassette only. Here, we provide descriptions of the required plasmids for CRISPR-Cas9-mediated integration into the AAVS1 locus in cultured human cells, as well as their transfection and subsequent selection of resulting knock-in populations. Finally, we propose a straightforward approach for the generation and validation of knock-in cell lines (Fig. 1). The NHEJ-mediated knock-in occurs at a genomic safe harbor and all components necessary for selection and amber suppression are contained in the integrated sequence. Therefore, indels at the repaired DSB junctions do not affect the function of the insert. AAVS1 pAS knock-in cell lines can be used

Fig. 1 Workflow of CRISPR-Cas9-mediated generation of amber suppression cell lines. Step 1: Cells are cotransfected with a pAS plasmid (template to deliver the amber suppression components), a sgRNA/Cas9 plasmid with pAS-targeting sgRNA sequence, and a sgRNA/Cas9 plasmid targeting the AAVS1 locus. Step 2: Cells are selected for successful integration via a resistance marker in the knocked-in pAS plasmid. PCR of extracted genomic DNA validates integration. Integrant cell lines can be further validated by immunoblotting. Step 3: The validated pAS knock-in cell lines can be used for expression of amber suppression proteins

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for all applications of ncAA-labeled protein synthesis via amber suppression, such as amber mutant screening, protein production and purification, and click chemistry-based imaging applications.

2

Materials 1. Plasmids for S. pyogenes Cas9 and sgRNA expression as well as pAS plasmids for amber suppression (if necessary, generate new plasmid by molecular cloning; detailed protocols are published [1, 10]). The plasmids for sgRNA/Cas9 expression are based on pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988) from Feng Zhang’s lab [9]. The puromycin resistance marker was removed by EcoRI restriction digest and religation. The pAS (amber suppression) plasmids for PylT/RS expression and GOI receiving plasmid (NheI/NotI insertion) as well as GFP150TAG reporter plasmids are available from Addgene (Tables 1 and 2). 2. HEK293T cells (e.g., ATCC #CRL-3216) and cell culture equipment (see Note 1). 3. DMEM supplemented with 10% v/v FBS for culturing HEK293T cells, or appropriate medium for other cell types. 4. Cell dissociation agent, e.g., trypsin, AccutaseTM (ICT), or TrypLETM (Life Technologies).

Table 1 CRISPR-Cas9 plasmids Addgene #

Name

Guide sequence

Citation

193309

pX459_gRNA AAVS1_hspCas9

GTCCCCTCCACCCCACAGTG

This protocol

193311

pX459_gRNA pAS single_hspCas9 GTCACGACGTTGTAAAACGA

This protocol

193312

pX459_gRNA pAS dual_hspCas9 GAGGGACGTAATTACATCCC

This protocol

Table 2 pAS plasmids Addgene # Name

Selection E. coli Selection

Citation

140008

pAS (GOI receiving plasmid)

Ampicillin

Blasticidin

[2]

140009

pAS_4xMma PylT_FLAG-Mma PylRS

Ampicillin

Puromycin [2]

140015

pAS_4xMma PylT_GFP150TAG

Ampicillin

Blasticidin

140023

pAS_4xMma PylT_FLAG-Mma PylRS AF Ampicillin

Puromycin [2]

193313

pAS1_sfGFP150K

Blasticidin

Ampicillin

[2]

This protocol

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Table 3 Genotyping primers

ID

Name

Sequence

Amplicon Amplicon + primer 1 + primer 2

Primer 1

AAVS_KI_L1

TGGGGGTGTGTCACCAGATA

–

400 bp

Primer 2

AAVS_KI_R1

GTGGATTCGGGTCACCTCTC

400 bp

–

Primer 3s

pAS single_KI_L1

GCCTCTTCGCTATTACGCCAG

210 bp

360 bp

Primer 4s

pAS single_KI_R1

GTCCTCCACGGGTTCAAAAAC

190 bp

330 bp

Primer 3d

pAS dual_KI_L1

CCAGGTTTAGCCCCGGAATTG

390 bp

530 bp

Primer 4d

pAS dual_KI_R1

GCTTCTCGCTGCTCTTTGAGC

300 bp

440 bp

5. Reduced serum medium such as Opti-MEMTM I ReducedSerum Medium (Thermo Fisher Scientific). 6. 1 mg/mL polyethyleneimine (PEI) transfection solution (see Note 2). 7. A high-speed tabletop centrifuge. 8. 10 mg/mL puromycin stock solution in sterile deionized water and 20 mg/mL blasticidin stock solution in cell culture media. Store stock solutions at -20 °C. 9. Extraction kit for genomic DNA (see Note 3), heat block. 10. PCR machine, high-speed temperature-stable DNA polymerase, e.g., DreamTaqTM (Thermo Fisher Scientific). 11. Primer for PCR validation of knock-in at the AAVS1 locus (see Table 3). 12. Agarose gel electrophoresis equipment. 13. Equipment and reagents for SDS-PAGE and immunoblotting. 14. ncAAs. For example, 0.2–0.5 mM cyclopropene lysine (CpK) (SiChem, SC-8017; CAS:1610703–09-7) in the cell medium for amber suppression with wild-type Mma PylRS and 0.1 mM trans-cyclooctene lysine (TCO*K) (SiChem: SC-8008; CAS:1801936-26-4) in the cell medium for amber suppression with Mma PylRS AF. Working stocks of ncAAs are prepared at 100 mM in 0.2 M NaOH, 15% (w/w) DMSO. 15. Antibodies: e.g., mouse monoclonal anti-FLAG HRP-coupled (Sigma, #A8592); GAPDH (chicken polyclonal, Millipore, #AB2302); anti-chicken IgY (H+L) HRP (Invitrogen, #A16054). 16. Flow cytometer.

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Methods

3.1 Transfection of Adherent Human Cell Lines

The pAS-based vector is introduced into the cells together with two sgRNA/Cas9 vectors, one targeting the human AAVS1 locus (pX459_gRNA AAVS1_hspCas9) and the other targeting the pAS plasmid (either pX459_gRNA pAS single_hspCas9 or pX459_gRNA pAS dual_hspCas9; see Fig. 2, Table 1 and Note 4). We recommend using the latter, “dual-cut” sgRNA, to introduce two cuts in the insulator regions flanking the pAS cassette, hence inserting only the relevant part of the plasmid (see Note 5). A ratio of 1:1:1 was used to generate the cell lines for the example data in Figs. 3 and 4. A pAS GFP control vector (e.g., pAS_sfGFP150K; Addgene #193313) can be used as a positive control. This plasmid, combined with pX459_gRNA AAVS1_Cas9 and the sgRNA expression plasmids of choice, allows the generation of a GFP knock-in cell line that can be easily assessed for integration, selection, and validation. After antibiotic selection, polyclonal cell lines can be used for most applications. In Subheading 3.3 we detail the isolation of individual clonal populations. 1. Seed 4 × 105 HEK293T cells into the wells of a 6-well plate, one well per transfection. Culture the cells at 37 °C with 5% CO2 atmosphere for 24 h prior to transfection. 2. Prepare 3 μg of total DNA at 1:1:1 ratio in 150 μL of Opti-MEM. 3. Add 9 μL of 1 mg/mL PEI. 4. Vortex 5–10 s to mix.

Fig. 2 Targeting CRISPR-Cas9 to pAS. Schematic illustration of the two sgRNA targeting strategies described. (a) The “single-cut” sgRNA is designed to induce CRISPR-Cas9-mediated incision outside of the amber suppression expression cassette. DNA repair mechanisms lead to the knock-in of linearized pAS. The “dualcut” sgRNA targets the inverted repeats flanking the amber suppression expression cassette. In both strategies, integrants with the correct pAS fragment are selected via antibiotic markers. (b) Sequences targeted in pAS. The sequence the sgRNA anneals to is underlined and color-coded as in (a). The NGG PAM sequences immediately 3′ of the targeted sequence are indicated (in orange) as well as the expected site of the DSB (dashed red line)

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Fig. 3 Confirming pAS genomic insertion by PCR. (a) Scheme illustrating the possible orientations of integration at the AAVS1 locus and genotyping primer binding sites.(b) agarose gel of PCR performed on genomic DNA isolated after CRISPR-Cas9 knock-in using the “single-cut” strategy with pAS_4xMmaPylT_ MmaPylRS (Mma PylT/R, Addgene #140009), comparison to piggyBac integration of the same plasmid. Triangles representing the amplification primers are color-coded as in (a). See also Table 3

Fig. 4 Example data. HEK293T knock-in cell lines generated with pAS_4xMmaPylT_MmaPylRS using sgRNA pAS “single-cut” or sgRNA pAS “dual-cut” strategies. (a) Immunostaining for expression of Mma PylRS. Cell lysates prepared from equal cell number aliquots of HEK293T parental and Mma PylRS knock-in cells were separated by SDS-PAGE and immunostained for FLAG-tagged PylRS and GAPDH. (b and c) The cell lines were transiently transfected with pAS_4xMmaPylT_GFP150TAG and cultured for 24 h in medium supplemented with 0.2 mM cyclopropene lysine (+CpK) or in normal DMEM (-ncAA). (b) Fluorescence imaging on a ZOE imager (BioRad). Scale bar indicates 100 μm. (c) Intracellular GFP fluorescence measured by flow cytometry

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5. Incubate at room temperature 20–30 min for formation of transfection complexes. 6. Add 2.5 mL fresh medium to the transfection mix (may be supplemented with penicillin and streptomycin). 7. Aspirate medium from the wells to be transfected and replace it with the prepared medium with transfection mix. 3.2 Selection of Polyclonal Pools

1. Forty-eight hours after transfection, split the cells into three different wells of a new 6-well plate. Add various concentrations of selection antibiotics to find the strongest tolerated selection condition. For example, use 1 μg/mL, 2 μg/mL, and 5 μg/mL of puromycin to select for integration of a PylT/PylRS cassette. For selection of a blasticidin resistance marker (e.g., GFP control plasmid pAS_GFP150K, Addgene# 193313), appropriate concentrations for HEK293T cells are 200 μg/mL, 500 μg/mL, and 1 mg/mL. 2. Maintain and monitor selection for 7 days to ensure stable integration and loss of transient vectors. Renew selection media every 2–4 days. At low drug concentration, it may be necessary to split the cells grown too densely. 3. Surviving cells can be reselected at higher drug concentrations, but it is advisable to first expand the initially selected pool enough to freeze an aliquot. 4. Expand the cells surviving the most stringent antibiotic selection.

3.3 Selection of Individual Clones

1. Split cells 48 h after transfection and dilute to 3 × 10 mL with three different concentrations of selection drug, seeding each into one 10 cm dish. 2. Maintain and monitor selection for 7 days to ensure stable integration and loss of transient vectors by renewing selection every 2–4 days. 3. When colonies become visible, aspirate the medium and carefully collect single clonal colonies using a pipette with a small amount of trypsin. Transfer the cells into wells of a 12-well plate with fresh medium and expand.

3.4 Validation of AAVS1 Knock-In Cell Line

Successful integration of any pAS vector into the AAVS1 locus can be validated by PCR with genomic DNA extracted from the generated cell line and sets of primers annealing in the AAVS1 locus and pAS sequence. See Fig. 3 for exemplary results. Note that in a polyclonal cell population detection of all possible amplification products is expected, as knock-in occurs with random orientation at each targeted AAVS1 allele. Expression of the knocked-in PylRS can be assessed by immunoblotting – the pAS plasmids for MmaPylRS and Mma PylRS AF encode an N-terminal FLAG-tag on the respective PylRS (Table 2).

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1. Collect 0.5–1 × 106 cells (e.g., when splitting) and pellet 5 min at 300×g. 2. Wash the cell pellet once with PBS. 3. Extract genomic DNA using a commercially available kit (see also Note 3). 4. Set up DreamTaq (or equivalent polymerase) reactions with primers 1 + 2 (amplifies 400 bp in the AAVS1 locus flanking the sequence target by the sgRNA) and with combinations of one of the outside primers (1 and 2) with one of the primers annealing within pAS (primers 3 and 4). Depending on the strategy and sgRNA used to linearize the pAS plasmid, either primers 3s and 4s (“single-cut”) or primers 3d and 4d (“dualcut”) should be used. These primers are universal to all pAS plasmids and anneal outside of the gene expression cassette. Refer to Table 3 for expected amplicon sizes. 5. Run PCR reactions as appropriate for the DNA polymerase used and analyze on a 2% agarose gel. 3.5 Implementing AAVS1 Knock-In Cell Lines in Amber Suppression Experiments

Once a stable cell line is generated in HEK293T cells, it can be used for transient transfection. A stable PylRS transgenic cell line can be used for routine screening of new amber suppression GOI plasmids, e.g., optimizing amber codon position in the sequence, with the ease of single-vector transient transfection. Exemplary results are shown in Fig. 4. 1. Seed 1.5–2 × 105 validated AAVS1 knock-in cells for each planned transfection in a 24-well plate 24 h prior to transfection. Ideally, include an extra well for each plasmid to be transfected and culture without ncAA as negative control. 2. Prepare 1 μg of DNA per well. 3. Add 50 μL of OptiMEM per 1 μg of DNA. 4. Add 3 μL of PEI per 1 μg of DNA and vortex for 5–10 s. 5. Incubate at room temperature for 20–30 min for formation of transfection complexes. 6. During the incubation, prepare 2× final concentration ncAA in medium (0.5 mL per transfection). 7. Add 450 μL of fresh medium to the transfection mix (may be supplemented with penicillin and streptomycin). 8. Aspirate medium from the wells to be transfected and replace it with the prepared medium with transfection mix. 9. Add either 0.5 mL of 2× ncAA (amber suppression) or 0.5 mL of medium (negative control) to the cells with transfection mix. 10. Assess amber suppression-mediated expression of the protein of interest 24–48 h post-transfection with the appropriate

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detection method. If a fluorescent amber suppression reporter was included as positive control, it will be detectable after 24 h and can be used to assess transfection efficiency. 3.6 Generating Full Stable Amber Suppression Cell Lines from PylRS Knock-In Cell Lines

4

AAVS1 knock-in stables expressing PylRS provide a defined starting point for generating full stable amber suppression cell lines by subsequent integration of the PylT/GOI plasmid via piggyBac transgenesis. To this end, AAVS1 knock-in cell lines generated as described above with the pX459_gRNA pAS dual_hspCas9 plasmid can be further derivatized following the published protocol for generating full stable amber suppression cell lines [1] starting with Subheading 3.2. (see also Note 6).

Notes 1. The protocol is optimized for HEK293T cells, as they are efficiently transfected and allow transient introduction of additional plasmids after stable integration. Use of HEK293T cells precludes the use of geneticin/neomycin as a selectable marker, since this resistance was used to introduce the SV40 large T-antigen. Care should be taken to periodically reselect stable cell lines generated in genomically unstable cells (such as HEK293T) to prevent the spontaneous loss of the transgene. When using different cells, it is advisable to determine sensitivity to the antibiotic to be used. 2. Other lipofection agents can be used as well. When using PEI for transfection, it is important to use the specific product from Polysciences (polyethylenimine 25 kDa linear, cat. # 23966-2). The length and branching may vary on other products. The DNA/PEI ratio should be optimized for every batch of PEI. A 1:3 DNA/PEI ratio usually yields good results. 3. Genomic DNA can also be extracted following classic isolation protocols that typically yield enough material for verification PCR. Such protocols are published in standard laboratory manuals (e.g., [12]). 4. Plasmids pX459-gRNA AAVS1_Cas9, pX459-gRNA pAS single_Cas9, and pX459-gRNA pAS dual_Cas9 are available from Addgene (see Table 1). They were generated by ligation of annealed, phosphorylated oligos to BbsI-digested pSpCas9 (BB)-2A-Puro (PX459) V2.0 (Addgene #62988) following a general protocol deposited by Feng Zhang’s lab to Addgene. Following these procedures, sgRNAs targeting other genomic loci or donor plasmids can be generated. The original pX459 plasmid carries a puromycin resistance marker flanked by EcoRI sites. To avoid the retention of sgRNA/Cas9 plasmids selected with puromycin, we religated the plasmids after EcoRI digestion to remove the puromycin selection marker (see Table 2).

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5. In case the parental cell line used for this protocol contains prior piggyBac-mediated insertions, please note that the “dualcut” sgRNA expressed from pX459_gRNA pAS dual_hspCas9 will also target the insulator regions of these integrants. Therefore, the pX459_gRNA pAS single_hspCas9 plasmid should be used to avoid cutting and potentially excising prior piggyBac integrants. 6. AAVS knock-in cell lines generated with the pX459_gRNA pAS single_hspCas9 plasmid contain the entire pAS plasmid sequence including the bacterial origin of replication, bacterial selection marker, and piggyBac inverted terminal repeats. The introduction of piggyBac transposase into such cell lines will potentially excise the PylT/PylRS cassette from the AAVS locus. Stable cell lines generated with the pX459_gRNA pAS single_hspCas9 plasmid are for this reason incompatible with subsequent piggyBac transgenesis.

Acknowledgements We thank the members of the Els€asser lab for helpful comments and discussion. References 1. Els€asser SJ (2018) Generation of stable amber suppression cell lines. Methods Mol Biol 1728: 237–245. https://doi.org/10.1007/978-14939-7574-7_15 2. Meineke B, Heimg€artner J, Lafranchi L, Els€asser SJ (2018) Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells. ACS Chem Biol 13: 3087–3096. https://doi.org/10.1021/ acschembio.8b00571 3. Lafranchi L, Schlesinger D, Kimler KJ, Els€asser SJ (2020) Universal single-residue terminal labels for fluorescent live cell imaging of microproteins. J Am Chem Soc 142:20080–20087. https://doi.org/10.1021/jacs.0c09574 4. Meineke B, Heimg€artner J, Eirich J, Landreh M, Els€asser SJ (2020) Site-specific incorporation of two ncAAs for two-color bioorthogonal labeling and crosslinking of proteins on live mammalian cells. Cell Rep 31: 107811. https://doi.org/10.1016/j.celrep. 2020.107811 5. Els€asser SJ, Ernst RJ, Walker OS, Chin JW (2016) Genetic code expansion in stable cell lines enables encoded chromatin modification.

Nat Methods 13:158–164. https://doi.org/ 10.1038/nmeth.3701 6. Bartoschek MD, Ugur E, Nguyen T-A, Rodschinka G, Wierer M, Lang K, Bultmann S (2021) Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells. Nucleic Acids Res 49:e62. https://doi.org/10. 1093/nar/gkab132 7. Smith JR, Maguire S, Davis LA, Alexander M, Yang F, Chandran S, ffrench-Constant C, Pedersen RA (2008) Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells 26:496–504. https://doi.org/10. 1634/stemcells.2007-0039 8. Dubois VP, Zotova D, Parkins KM, Swick C, Hamilton AM, Kelly JJ, Ronald JA (2018) Safe harbor targeted CRISPR-Cas9 tools for molecular-genetic imaging of cells in living subjects. CRISPR J 1:440–449. https://doi. org/10.1089/crispr.2018.0030 9. Jiang F, Doudna JA (2017) CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys 46:505–529. https://doi.org/10.1146/ annurev-biophys-062215-010822

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10. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308. https://doi.org/10.1038/ nprot.2013.143 11. He X, Tan C, Wang F, Wang Y, Zhou R, Cui D, You W, Zhao H, Ren J, Feng B (2016) Knockin of large reporter genes in human cells via

CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res 44:e85. https://doi.org/10.1093/nar/ gkw064 12. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York

Chapter 13 Genetic Code Expansion in Mammalian Cells Through Quadruplet Codon Decoding Yan Chen, Tianyu Gao, Xinyuan He, Wei Niu, and Jiantao Guo Abstract Genetic code expansion enables the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins both in vitro and in vivo. In addition to a widely applied nonsense suppression strategy, the use of quadruplet codons could further expand the genetic code. A general approach to genetically incorporate ncAAs in response to quadruplet codons is achieved by utilizing an engineered aminoacyl-tRNA synthetase (aaRS) together with a tRNA variant containing an expanded anticodon loop. Here we provide a protocol to decode quadruplet UAGA codon with a ncAA in mammalian cells. We also describe microscopy imaging and flow cytometry analysis of ncAA mutagenesis in response to quadruplet codons. Key words Non-canonical amino acid, Unnatural amino acid, Genetic code expansion, Quadruplet codon, Four-base codon

1

Introduction The genetic code expansion approach has been used to incorporate noncanonical amino acids (ncAAs) into protein both in vitro and in vivo [1–3]. By introducing novel functional and structural properties to proteins, ncAA mutagenesis has proven to be a useful toolkit in biological studies and medicinal applications. The amber nonsense codon (UAG) is commonly used to encode ncAAs. Exploring additional codons to encode ncAAs has been one of the focuses in the field of genetic code expansion as the number of naturally existing nonsense codons is limited. To this end, quadruplet codons are emerging as excellent choices to further expand the genetic code (Fig. 1a) [4, 5]. Indeed, quadruplet codons have been applied for this purpose in bacteria [6–14], mammalian cells [15–17], and Caenorhabditis elegans [18]. However, the low decoding efficiency hampers broad applications of quadruplet codon decoding in ncAA mutagenesis, especially in mammalian cells. We [11, 15] and others [9] demonstrated that

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_13, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Noncanonical amino acid incorporation in response to quadruplet codons. (a) Schematic of quadruplet codon decoding. The aminoacyl-tRNA synthetase (aaRS) charges a ncAA onto the quadruplet codon decoding tRNA that contains an extended anticodon loop. The charged tRNA decodes the quadruplet codon in the gene of interest. (b) Structure of Nε-(tert-butyloxy-carbonyl)-L-lysine (BocLys)

quadruplet codon decoding efficiency could be significantly enhanced by engineering the anticodon stem loop of pyrrolysyltRNA (PylT). In our previous reports [15, 19], directed evolution of Methanosarcina barkeri PylT (MbPylT) was carried out in Escherichia coli (E. coli). The identified MbPylT variants, tRNAUCCU and tRNAUCUA, could be directly utilized to decode AGGA and UAGA codon in mammalian cells, respectively [11, 15]. To further improve decoding efficiency, a hybrid tRNA variant, tRNAUCUAM15, that contains an optimized scaffold for eukaryotic translation machinery was also generated [18, 20]. In the following sections, we provide a protocol for the genetic incorporation of ncAA in response to a quadruplet UAGA codon in mammalian cells using an orthogonal MbPylRS/PylT pair. Three quadruplet codon decoding tRNAs are used, including wt-tRNAUCUA (MbPylT with an apparent UCUA anticodon), tRNAUCUA-1 (with additional mutations in anticodon stem-loop), and tRNAUCUA-M15 (a hybrid tRNA). An evolved PylRS, BocLysRS, that specifically aminoacylate tRNA with Nε-(tert-butyloxy-carbonyl)L-lysine (BocLys, Fig. 1b) is used. To demonstrate ncAA incorporation, an enhanced green fluorescent protein (EGFP) gene with an UAGA codon for the 40th amino acid residue is used as a reporter. Details are provided on plasmid design, protocols for transfecting human embryonic kidney (HEK) 293T cells, microscopy imaging, and flow cytometry analysis.

2

Materials

2.1 Noncanonical Amino Acid

2.2

Plasmids

500 mM Nε-(tert-butyloxy-carbonyl)-L-lysine (BocLys, Fig. 1b): Dissolve 0.123 g of BocLys in 1 mL of 1 M NaOH. Store the 500 mM stock solution at -20 °C (see Note 1). 1. Plasmid pcDNA3.1 vector is used as the backbone to construct plasmids. Primer sequence is shown in Table 1 (see Note 2).

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Table 1 Primer sequence used in this protocol Name

Sequence (5′ to 3′)

Plasmid

tRNA-F tRNA-R

gcttgaccgacaattgcagcacagaaaaacggaaacccc attcttcatgcaattgtcgggcagg

pBocLysRS-wt-tRNAUCUA/ tRNAUCUA-1

tRNA-M15-F tRNA-M15-R M15-FspI-F M15-SpeI-R

aggattagaagcccgttcggtctccctgaccaggtttccggt gggcttctaatcctgttcagccgggttcgattcccggggt gttaatagtttgcgcaacgttgttg tattaataactagtcaataatcaat

pBocLysRS-tRNAUCUA-M15

40-F 40-R 40-NheI-F 40-XhoI-F

atgccacctagaggcaagct agcttgcctctaggtggcat aggcgtgtacggtgggaggtct gatggctggcaactagaagg

pEGFP-40UAGA

Fig. 2 Sequence of tRNAUCUA variants and plasmid design. (a) Sequence of three tRNAUCUA variants that are used in this study. The anticodon UCUA, which recognizes the UAGA codon, is labeled in bold with underline. Mutations in tRNAUCUA-1 and tRNAUCUA-M15 are highlighted in red color. (b) Plasmid design of pBocLysRStRNAUCUA variants and pEGFP-40UAGA

2. Plasmid pBocLysRS-tRNAUCUA variants: The encoding gene of wt-tRNAUCUA or tRNAUCUA-1 (Fig. 2a) is PCR amplified, digested with MfeI, and ligated into the plasmid pCMVBocLysRS (see Note 3) [15] that is treated with the same set of restriction enzymes. The wt-tRNAUCUA is derived from wild-type MbPylT by changing its anticodon sequence from CUA into UCUA. The tRNAUCUA-1 mutant is obtained through directed evolution in E. coli (Fig. 2a). Reported mutations [18, 20] in the encoding gene of tRNAUCUA-M15 (Fig. 2a) were introduced using overlapping PCR. The PCR products are digested with FspI and SpeI and ligated into the vector plasmid pBocLysRS-tRNAUCUA-1 that was treated with

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the same set of restriction enzymes. These plasmids also encode BocLysRS that can specifically charge quadruplet codon decoding tRNAs with BocLys. The expression of BocLysRS and tRNAs is controlled by a nonregulated CMV promoter and a human U6 promoter (Fig. 2b), respectively. 3. Plasmid pEGFP-40UAGA (Fig. 2b): Overlapping PCR is used to introduce a UAGA codon into the EGFP-encoding gene at position Y40. The PCR product is digested with NheI and XhoI and ligated into the plasmid pEGFP-wt [15] that is treated with the same set of restriction enzymes. The expression of EGFP-40UAGA is under the control of a nonregulated CMV promoter (Fig. 2b). 2.3

Cell Culturing

1. Human embryonic kidney (HEK) 293T cells. 2. DMEM, high glucose, pyruvate. 3. 0.05% trypsin-EDTA, phenol red. 4. Fetal bovine serum (FBS): Inactivate FBS in a 56 °C water bath for 30 min before preparing the 10% FBS DMEM. 5. 10% FBS DMEM: Mix 450 mL of DMEM with 50 mL of heatinactivated FBS and sterilize it by filtration with a 500 mL PES filter unit. 6. Dimethyl sulfoxide (DMSO). 7. Cell freezing system. 8. 24-well cell culture plates. 9. T25 cell culture flasks. 10. Lipofectamine 2000. 11. 4% formaldehyde solution: Dilute 50 mL of 40% formaldehyde solution in 450 mL of DPBS. 12. 0.2 μm sterile syringe filter. 13. 96-well microplate.

2.4 Confocal Imaging and Flow Cytometry Analysis

3 3.1

1. Fluorescence microscope. 2. Confocal microscope. 3. Flow cytometer (e.g., Beckman Coulter CytoFLEX LX).

Methods Cell Culture

1. To revive HEK293T cells, thaw 1 mL of frozen cells in a 37 °C water bath for 1 min. 2. Mix thawed cells with 4 mL of pre-warmed 10% FBS DMEM in a T25 flask. Grow cells at 37 °C in a humidified atmosphere of 5% CO2 (see Note 4).

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3. Passage cells at around 80% confluency to a new T25 flask. To do this, remove culture medium and add 2 mL of 0.05% trypsin-EDTA. Incubate cells at 37 °C for 3 min. 4. When most cells are detached, add 3 mL of 10% FBS DMEM to inhibit trypsin. Gently resuspend cells by pipetting and transfer 0.5 mL of the cell suspension to 4.5 mL of pre-warmed 10% FBS DMEM. Incubate cells at 37 °C in an incubator with 5% CO2 (see Note 5). 5. To prepare HEK293T cell stock, detach cells by following steps 3 and 4. Mix 900 μL of cell suspension with 100 μL of DMSO and transfer cells to a cryogenic vial. Slowly freeze the cells in a cell freezing system at -80 °C overnight. Transfer the frozen cells to liquid nitrogen the next day (see Note 6). 3.2

Transfection

1. One day before transfection, detach HEK 293T cells from a T25 flask with 2 mL of 0.05% trypsin-EDTA and add 3 mL of 10% FBS DMEM. Properly dilute the cell suspension and plate 0.5–2 × 105 HEK 293T cells in 500 μL of 10% FBS DMEM in a 24-well cell culture plate. Antibiotics should not be added during the transfection. Incubate cells at 37 °C in an incubator with 5% CO2 until cells reach 70–80% of confluency (see Note 7). 2. Prepare 10% FBS DMEM supplemented with BocLys before transfection. To make 1 mL of 10% FBS DMEM supplemented with 5 mM BocLys, mix 10 μL of 500 mM BocLys with 980 μL of 10% FBS DMEM, and adjust pH by adding 10 μL of 1 M HCl. Sterilize the solution by filtering through a 0.2 μm filter. 3. When cells reach 70–80% of confluency, replace culture medium with 500 μL fresh and warm 10% FBS DMEM supplemented with 5 mM BocLys (see step 2). In control wells, culture medium is replaced with 500 μL of fresh and warm 10% FBS DMEM. Place culture plate back to the incubator while preparing the transfection reagent. 4. Prepare DNA dilution by mixing 50 μL of DMEM (no FBS) with 0.8 μg of pBocLysRS-tRNAUCUA variant of interest and 0.8 μg of pEGFP-40UAGA. Incubate the DNA mixture at room temperature for 5 min (see Note 8). 5. In parallel to step 4, prepare Lipofectamine dilution by mixing 48 μL of DMEM (no FBS) with 2 μL of Lipofectamine 2000. Incubate the mixture at room temperature for 5 min. 6. Mix gently the DNA dilution prepared in step 4 and the Lipofectamine dilution prepared in step 5. Incubate at room temperature for at least 20 min (see Note 9). 7. Add 100 μL of the mixture prepared in step 6 to each well. Mix gently by rocking the plate in one direction. Incubate cells at

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Table 2 Troubleshooting guidance Problem

Possible causes

Low transfection efficiency

Unhealthy cell

Low ncAA incorporation efficiency

Low transfection efficiency Low quadruplet codon decoding efficiency Bad ncAA solution

Possible solutions

Use healthy cell and check cell fitness with microscopy before transfection Cell confluency is too low Seed cell with proper dilution to achieve 70–80% or too high confluency at transfection Low quality of plasmid Prepare plasmid with higher purity and concentration Bad transfection reagent Purchase new Lipofectamine 2000 or Lipofectamine 3000 (more efficient) Optimize the transfection process Adjust the expression level of aaRS/tRNA or engineer the aaRS/tRNA pair Prepare fresh ncAA solution and adjust pH of culture media to neutral before transfection

37 °C in an incubator with 5% CO2. The EGFP expression can be visualized under a fluorescent microscope 12 to 24 h after transfection. Cells are ready for microscopy imaging/flow cytometry analysis 24 to 48 h after transfection (see Note 10 and Table 2). 3.3 Sample Preparation for Confocal/Fluorescence Imaging

1. When cells are ready for imaging (24–48 h after transfection), remove culture medium and wash cells with 500 μL of DPBS. Live cells can be directly imaged (see Note 11). For imaging of fixed cells, go to step 2. 2. Remove DPBS and add 500 μL of 4% formaldehyde solution. Incubate the culture plate at room temperature for 20 min. 3. Remove 4% formaldehyde and wash cells with 500 μL of DPBS twice. 4. Visualize cell samples using a fluorescence microscope (see Note 11). The samples are excited at 488 nm to acquire EGFP fluorescence at 530/25 nm. At least three images are acquired at different locations for each sample (Fig. 3a, c).

3.4 Flow Cytometry Analysis Sample Preparation

1. When cells are ready for flow cytometry analysis (24–48 h after transfection), remove culture medium (see Note 12). 2. Detach cells by adding 200 μL of 0.05% trypsin-EDTA and incubate the culture plate at 37 °C. Once cells are detached (about 5 min of incubation), add 300 μL of 10% FBS DMEM to inhibit trypsin. 3. Resuspend cells by pipetting and transfer cells to a 1.5 mL Eppendorf tube. Harvest cells by centrifuging at 300 g and 4 °C for 5 min, remove supernatant, and resuspend cells in 500 μL of cold DPBS.

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Fig. 3 Analysis of BocLys incorporation in response to UAGA codon. (a) Confocal images of HEK293T cells co-transfected with pBocLysRS-tRNAUCUA (or pBocLysRS-tRNAUCUA-1) and pEGFP-40UAGA in the presence or absence of 5 mM BocLys. (b) Flow cytometry analysis of confocal images of HEK293T cells co-transfected with pBocLysRS-tRNAUCUA (or pBocLysRS-tRNAUCUA-1) and pEGFP-40UAGA in the presence or absence of 5 mM BocLys. Normalized fluorescence is calculated by multiplying the mean fluorescence intensity by the percentage of fluorescent cells. Each data point is the average of triplet measurements with standard deviation. (c) Fluorescence images of HEK293T cells co-transfected with pBocLysRS-tRNAUCUA-1 (or pBocLysRS-tRNAUCUA-M15) and pEGFP-40UAGA in the presence or absence of 5 mM BocLys

4. Centrifuge at 300 g and 4 °C for 5 min to remove the supernatant and resuspend in 200 μL of 4% formaldehyde for fixation. Incubate cells at room temperature for 20 min. 5. Centrifuge at 300 g and 4 °C for 5 min to remove supernatant and wash with cells with 300 μL of cold DPBS. Repeat the DPBS wash one more time. 6. Resuspend cells in 300 μL of cold DPBS by pipetting and transfer 200 μL of cell samples to a clear 96-well plate. 7. Analyze cell samples using Beckman Coulter CytoFLEX flow cytometer (see Note 13). Cell samples are excited at 488 nm to measure EGFP fluorescence at 525/40 nm (BLU1). Positive

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(cells transfected with pEGFP-wt) and negative (cells only) controls are used to adjust EGFP detection sensitivity. For each sample, 3 × 105 cells are analyzed and/or collected using CytExpert software. Two parameters are recorded: percentage of fluorescent cells and mean fluorescence intensity. Normalized fluorescence is calculated by multiplying the mean fluorescence intensity by the percentage of fluorescent cells (Fig. 3b).

4

Notes 1. Some ncAAs are soluble at neutral pH or more stable under acidic conditions. Change the preparation process according to the nature of ncAAs. Lower the concentration of stock solutions to 100 mM if necessary. 2. For mammalian expression of tRNA, the 3′-CCA is posttranscriptionally added. Therefore, the 3′-CCA of tRNA is omitted during plasmid construction. 3. The following is the DNA sequence of BocLysRS. atggataaaaaaccattagatgttttaatatctgcgaccgggctctggatgtccaggact ggcacgctccacaaaatcaagcaccatgaggtctcaagaagtaaaatatacattga aatggcgtgtggagaccatcttgttgtgaataattccaggagttgtagaacagcc agagcattcagacatcataagtacagaaaaacctgcaaacgatgtagggtttcgg acgaggatatcaataattttctcacaagatcaaccgaaagcaaaaacagtgtgaaa gttagggtagtttctgctccaaaggtcaaaaaagctatgccgaaatcagtttcaag ggctccgaagcctctggaaaattctgtttctgcaaaggcatcaacgaacacatcc agatctgtaccttcgcctgcaaaatcaactccaaattcgtctgttcccgcatcggct cctgctccttcacttacaagaagccagcttgatagggttgaggctctcttaagtcc agaggataaaatttctctaaatatggcaaagcctttcagggaacttgagcctgaac ttgtgacaagaagaaaaaacgattttcagcggctctataccaatgatagagaagac tacctcggtaaactcgaacgtgatattacgaaatttttcgtagaccggggttttctg gagataaagtctcctatccttattccggcggaatacgtggagagaatgggtattaa taatgatactgaactttcaaaacagatcttccgggtggataaaaatctctgcttgag gccaatgcttgccccgactctttacaactatctgcgaaaactcgataggattttacc aggcccaataaaaattttcgaagtcggaccttgttaccggaaagagtctgacggc aaagagcacctggaagaatttactatggtgaacttctgtcagatgggttcgggat gtactcgggaaaatcttgaagctctcatcaaagagtttctggactatctggaaatcgacttcgaaatcgtaggagattcctgtatggtctttggggatactcttgatataatg cacggggacctggagctttcttcggcagtcgtcgggccagtttctcttgatagag aatggggtattgacaaaccatggataggtgcaggttttggtcttgaacgcttgctcaaggttatgcacggctttaaaaacattaagagggcatcaaggtccgaatcttact ataatgggatttcaaccaatctgtaa 4. If the cell stock contains 10% DMSO, replace the culture medium after 24 h with 5 mL of 10% FBS DMEM to remove DMSO.

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5. Confluency and fitness of cells are critical to transfection. For cells revived from liquid nitrogen, passage three to four generations before transfection. 6. Frozen cells can be stored at -80 °C for up to 6 months. For long-term use, storage in liquid nitrogen is required. 7. Check cell fitness and confluency before transfection. Good fitness and proper confluency are critical to an efficient transfection. 8. The concentration of plasmids is important for efficient transfection. Adding a smaller volume of plasmids usually leads to higher transfection efficiency. 9. For different culture plates (e.g., 6-well or 96-well), prepare the DNA and Lipofectamine dilution by following the manufacturer’s instructions. 10. Cells transfected with pEGFP-wt plasmid can be used as the positive control. Cells only can be used as the negative control. 11. Prepare cell samples on the same day if imaging is preferred. Covered with alumni foil, the culture plate can be stored at 4 ° C for 24 h. 12. At least three replicates are required for each sample for flow cytometry analysis. 13. Prepare cell samples on the same day if flow cytometry analysis is preferred.

Acknowledgments This work was supported by the National Science Foundation (grant 1553041 to J.G.), National Institute of Health (grant 1R01GM138623 to J.G. and W.N.), and NIH National Institutes of General Medical Sciences (grant P20 GM113126 to J.G. and W.N.). References 1. Young DD, Schultz PG (2018) Playing with the molecules of life. ACS Chem Biol 13(4): 8 5 4 – 8 7 0 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 1 / acschembio.7b00974 2. Mukai T, Lajoie MJ, Englert M, Soll D (2017) Rewriting the genetic code. Annu Rev Microbiol 71:557–577. https://doi.org/10.1146/ annurev-micro-090816-093247 3. Chin JW (2017) Expanding and reprogramming the genetic code. Nature 550(7674): 5 3 – 6 0 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature24031

4. Wang K, Schmied WH, Chin JW (2012) Reprogramming the genetic code: from triplet to quadruplet codes. Angew Chem Int Ed 51(10):2288–2297. https://doi.org/10. 1002/anie.201105016 5. Guo J, Niu W (2022) Genetic code expansion through quadruplet codon decoding. J Mol Biol 434(8):167346. https://doi.org/10. 1016/j.jmb.2021.167346 6. Anderson JC, Wu N, Santoro SW, Lakshman V, King DS, Schultz PG (2004) An expanded genetic code with a functional quadruplet codon. Proc Natl Acad Sci U S A 101(20):

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7566–7571. https://doi.org/10.1073/pnas. 0401517101 7. Neumann H, Wang K, Davis L, Garcia-Alai M, Chin JW (2010) Encoding multiple unnatural amino acids via evolution of a quadrupletdecoding ribosome. Nature 464(7287): 4 4 1 – 4 4 4 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature08817 8. Rackham O, Chin JW (2005) A network of orthogonal ribosome·mRNA pairs. Nat Chem Biol 1(3):159–166. https://doi.org/10. 1038/nchembio719 9. Wang K, Sachdeva A, Cox DJ, Wilf NM, Lang K, Wallace S, Mehl RA, Chin JW (2014) Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat Chem 6(5): 393–403. https://doi.org/10.1038/nchem. 1919 10. Chatterjee A, Lajoie MJ, Xiao H, Church GM, Schultz PG (2014) A bacterial strain with a unique quadruplet codon specifying non-native amino acids. Chembiochem 15(12):1782–1786. https://doi.org/10. 1002/cbic.201402104 11. Wang N, Shang X, Cerny R, Niu W, Guo J (2016) Systematic evolution and study of UAGN decoding tRNAs in a genomically recoded bacteria. Sci Rep 6:21898. https:// doi.org/10.1038/srep21898 12. Hankore ED, Zhang L, Chen Y, Liu K, Niu W, Guo J (2019) Genetic incorporation of noncanonical amino acids using two mutually orthogonal quadruplet codons. ACS Synth Biol 8(5):1168–1174. https://doi.org/10. 1021/acssynbio.9b00051 13. Dunkelmann DL, Willis JCW, Beattie AT, Chin JW (2020) Engineered triply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids. Nat Chem 12(6):

535–544. https://doi.org/10.1038/s41557020-0472-x 14. Dunkelmann DL, Oehm SB, Beattie AT, Chin JW (2021) A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat Chem 13(11):1110–1117. https://doi.org/10.1038/s41557-02100764-5 15. Niu W, Schultz PG, Guo J (2013) An expanded genetic code in mammalian cells with a functional quadruplet codon. ACS Chem Biol 8(7):1640–1645. https://doi. org/10.1021/cb4001662 16. Chen Y, Wan Y, Wang N, Yuan Z, Niu W, Li Q, Guo J (2018) Controlling the replication of a Genomically recoded HIV-1 with a functional quadruplet codon in mammalian cells. ACS Synth Biol 7(6):1612–1617. https://doi.org/ 10.1021/acssynbio.8b00096 17. Mills EM, Barlow VL, Jones AT, Tsai Y-H (2021) Development of mammalian cell logic gates controlled by unnatural amino acids. Cell Rep Methods 1:100073 18. Xi Z, Davis L, Baxter K, Tynan A, Goutou A, Greiss S (2021) Using a quadruplet codon to expand the genetic code of an animal. bioRxiv. https://doi.org/10.1101/2021.07.17. 452788 19. Chen Y, Wan Y, Wang N, Yuan Z, Niu W, Li Q, Guo J (2018) Controlling the replication of a genomically recoded HIV-1 with a functional quadruplet codon in mammalian cells. ACS Synth Biol 7:1612–1617 20. Serfling R, Lorenz C, Etzel M, Schicht G, Boettke T, Moerl M, Coin I (2018) Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells. Nucleic Acids Res 46(1):1–10. https://doi.org/10.1093/nar/ gkx1156

Chapter 14 Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation Han Sun, Yang Huang, and Yu-Hsuan Tsai Abstract Site-specific modification of proteins has wide applications in probing and perturbing biological systems. A popular means to achieve such a modification on a target protein is through a reaction between bioorthogonal functionalities. Indeed, various bioorthogonal reactions have been developed, including a recently reported reaction between 1,2-aminothiol and ((alkylthio)(aryl)methylene)malononitrile (TAMM). Here, we describe the procedure that combines genetic code expansion and TAMM condensation for site-specific modification of cellular membrane proteins. The 1,2-aminothiol functionality is introduced through a genetically incorporated noncanonical amino acid to a model membrane protein on mammalian cells. Treatment of the cells with a fluorophore-TAMM conjugate leads to fluorescent labeling of the target protein. This method can be applied to modify different membrane proteins on live mammalian cells. Key words Genetic code expansion, Bioorthogonal reaction, Fluorescent labeling, 1,2-Aminothiol, TAMM condensation

1

Introduction Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems [1, 2]. A popular means to achieve such a modification on a target protein is through a reaction between bioorthogonal functionalities, which do not react with naturally occurring biomolecules but selectively react with each other under physiological conditions, consequently, eliminating concerns about side reactions and off-target effects. To date, many bioorthogonal reactions have been reported [3– 6]. Among these, a recently reported reaction involving 1,2-aminothiol and ((alkylthio)(aryl)methylene)malononitrile (TAMM) has a unique advantage that the chemical and physical properties of one reaction partner, TAMM, can be easily tuned without affecting the resulting product [7]. In addition, the small size of

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_14, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 (a) General reaction scheme of TAMM condensation. (b) Structure of CysK-OMe. (c) Structure of FITCPEG-TAMM

1,2-aminothiol warrants high atomic utilization efficiency and confers minimum perturbation to native proteins. In this chapter, we describe the procedure using TAMM condensation for site-specific fluorescent labeling of a membrane protein on live mammalian cells. Overexpressed mouse neuroligin-3 (mNlgn3) on HEK293T cells is used as the model protein. The 1,2-aminothiol functionality is introduced as a noncanonical amino acid, which bears a D-Cys on the Lys side chain and can be genetically incorporated into the target protein by Methanosarcina mazei PylRS [8] in response to the amber codon [9, 10]. In comparison to introducing 1,2-aminothiol functionality as a N-terminal Cys, using the non-canonical amino acid enables introduction of the functionality at any position of the target protein. For non-canonical amino acid incorporation, cells are treated with CysK-OMe (Fig. 1), the methyl ester derivative of the non-canonical amino acid, as the esterification enhances the cellular uptake of the amino acid [11]. Fluorescent labeling is accomplished with a fluorophore-TAMM conjugate, FITC-PEG-TAMM (Fig. 1). The PEG linker is employed to minimize the non-specific interaction between the conjugate and cell-surface biomolecules [12, 13]. In principle, this method can be applied to site-specific modification of different membrane proteins on live mammalian cells.

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Materials All commercial chemicals are of reagent grade or higher (see Note 1). All solution is prepared using ultrapure water unless otherwise indicated.

2.1

Plasmids

1. pPylRS [14]: This plasmid, available on Addgene (#140009), encodes M. mazei PylRS under the EF-1α promoter and four copies of Pyl tRNACUA under the human 7SK promoter, enabling site-specific incorporation of CysK in response to the amber codon. 2. pTAG-HA-mNlgn3: This plasmid encodes the model membrane protein, mouse neuroligin-3 (mNlgn3), with an amber (TAG) codon and HA tag after the signal peptide (amino acid 1-34). The plasmid is constructed by replacing the sequence between NheI and SalI sites of pPylRS with TAG-HA-mNlgn3 (see Note 2).

2.2 Cell Culture and Transfection

1. Fetal bovine serum (FBS). 2. DMEM high-glucose media. 3. 10,000 U/mL penicillin-streptomycin (P/S). 4. Complete culture medium: Add 50 mL of FBS and 5 mL of P/S into 445 mL of DMEM high-glucose media. 5. 0.25% trypsin. 6. Transfection reagent (e.g., PolyJet from SignaGen). 7. PBS. 8. 0.1% poly-L-lysine solution in ddH2O. 9. 100 mM CysK-OMe: Dissolve 24.5 mg of CysK-OMe • 2TFA in 500 μL of ddH2O.

2.3 In-Gel Fluorescence Analysis and Immunoblotting

1. Imaging buffer (see Note 3). 2. 10 mM FITC-PEG-TAMM: Dissolve 1 mg of FITC-PEGTAMM in 92 μL of DMSO. 3. 2 M DTT: Dissolve 100 mg of dithiothreitol in 324 μL of ddH2O. 4. Cell lysis buffer (see Note 4). 5. Loading buffer (see Note 5). 6. Precast 4–20% polyacrylamide gels. 7. 1 MOPS-SDS running buffer: 50 mM Tris base, 50 mM MOPS, 1 mM EDTA, 0.10% SDS. 8. 10 Transfer buffer: 25 mM Tris base, 192 mM glycine, 20% (v/v) methanol, pH 8.3.

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9. 0.45 μm nitrocellulose membrane. 10. Nonfat milk powder. 11. Mouse anti-HA antibody (ABclonal, AE008). 12. HRP-conjugated goat anti-mouse secondary antibody. 13. PBST: Add 0.5 mL of Tween-20 into 1000 mL of PBS. 2.4 Immunofluorescence Reagents

1. 4% paraformaldehyde fix solution (e.g., Beyotime, P0098). 2. DyLight 680 goat anti-mouse IgG secondary antibody (ThermoFisher, 35518). 3. DAPI solution (e.g., SolarBio, C0065). 4. Bovine albumin.

2.5

Equipment

1. Electrophoresis apparatus. 2. Biological safety cabinet. 3. CO2 incubator. 4. Orbital shaker. 5. Gel imager (e.g., Bio-Rad ChemiDoc Touch). 6. Confocal microscopy (e.g., ZEISS LSM980).

3 3.1

Methods Transfection

1. Plate 1  105 to 1.5  105 HEK293T cells per well to wells of a pre-treated 24-well plate (see Note 6). 2. When the monolayer cell density reaches ca. 70% confluency, change the media (see Note 7) and, if required, supplement cells with 1 mM CysK-OMe. 3. Prepare transfection mixture for each well by diluting 250 ng of pTAG-HA-mNlgn3 and 250 ng of pPylRS into 25 μL of serum-free DMEM. For each well, dilute 1.5 μL of PolyJet reagent into 25 μL of serum-free DMEM. Gently pipette up and down 3–4 times to mix. Add the diluted PolyJet reagent immediately to the diluted DNA solution all at once and incubate for 10–15 min at room temperature to allow PolyJet/ DNA complexes to form. Add the 50 μL of PolyJet/ DNA mixture drop-wise onto the medium in each well and homogenize the mixture by gently swirling the plate. 4. Incubate the cells at 37  C and 5% CO2 for 24 h.

3.2 In-Gel Fluorescence and Western Blot

1. Rinse cells with pre-warmed PBS (0.5 mL  3) and then incubated with 100 μM FITC-PEG-TAMM and 2 mM DTT (see Note 8) for 2 h at 37  C in imaging buffer (see Note 9).

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2. Rinse cells with imaging buffer (0.5 mL  4). 3. Remove imaging buffer, add 50 μL of cell lysis buffer to each well, and keep the plate on ice for 10 min. Bend a P200 tip and use it to scrape cells from the surface. Transfer all content from the well to a 1.5 mL tube. Centrifuge at 20,000  g and 4  C for 10 min. 4. Take 45 μL of supernatant to the new tube and add 15 μL of 4 loading buffer, gently mix by pipetting up and down. Heat the samples at 95  C for 10 min to denature protein. 5. Load 8 μL of each sample on a 4–20% polyacrylamide gel and electrophorese at 200 V for 60 min, by which time point the dye front should reach the bottom of the gel. 6. Image the gel on a gel imager using the fluorescence setting for FITC (or GFP). 7. Transfer the proteins onto a nitrocellulose membrane, and then block the membranes with 5% nonfat dry milk in PBST. 8. Incubate the membrane with mouse anti-HA primary antibody in 1:5000 dilution overnight at 4  C and thoroughly wash with PBST. Then incubating the membrane with goat anti-mouse HRP-conjugated secondary antibody in 1:5000 dilution for 1 h at room temperature and thoroughly wash with PBST. 9. Visualize the proteins with a chemiluminescence substrate on a gel imager (Fig. 2a).

Fig. 2 Fluorescent labeling of mNlgn3 bearing an N-terminal CysK and HA tag on HEK293T cells. (a) Representative gels for in-gel fluorescence and immunoblotting analysis. (b) Live-cell bioorthogonal labeling of CysK-HA-mNlgn3 with FITC-PEG-TAMM (green). The specificity of TAMM labeling is confirmed by immunofluorescence against HA (red). Nucleus are stained with DAPI (blue). Scale bars represent 5 μm

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3.3 Immunofluorescence

1. Transfer the transfected cells from one well of a 24-well plate into a pre-coated 35 mm glass-bottom dish (see Note 6). 2. Incubate the cells at 37  C and 5% CO2 for 8–12 h. 3. Rinse the cells with pre-warmed PBS (3  2 mL). 4. Incubate the cells with 100 μM FITC-PEG-TAMM and 2 mM DTT for 2 h at 37  C in imaging buffer (see Note 9). 5. Rinse the cells with imaging buffer (4  2 mL). 6. Fix the cells with 1 mL of 4% PFA at room temperature for 15 min and then rinse with PBS (3  2 mL). 7. Incubate the cells with 500 μL of 5% BSA in PBST at room temperature for 1 h. 8. Label the cells with mouse anti-HA primary antibody in 1:500 dilution for 1 h at room temperature and wash three times with PBS (5 min for each time). Then incubate the cells with DyLight 680 goat anti-mouse IgG secondary antibody in 1: 200 dilution for 1 h at room temperature and wash three times with PBS (5 min for each time). 9. Incubate the cells with DAPI solution at room temperature for 5 min. 10. Rinse the cells with 2 mL of PBS and image the sample on a confocal microscope (Fig. 2b).

4

Notes 1. CysK-OMe is available through custom synthesis from most suppliers of custom peptide synthesis service (e.g., ChinaPeptides, 100 mg for 500 USD). FITC-PEG-TAMM is also available through custom synthesis (e.g., WuXi AppTec, 10 mg for 3600 USD). 2. The DNA sequence of TAG-HA-mNlgn3 is shown below: ATGTGGCTGCAGCCCTCGCTGTCCCTGAGCCCCA CGCCCACAGTTGGCCGGAGCCTGTGCCTCACCCTGG GCTTCCTCAGTTTGGTGCTGAGGGCCAGTACCTAGC AGTATCCATATGATGTTCCAGATTATGCTGCCCCGGC ACCCACAGTCAATACTCACTTTGGGAAGCTAAGGGGT GCCAGAGTACCATTGCCCAGTGAAATCCTGGGTCCT GTGGACCAATACCTGGGGGTACCCTACGCAGCTCCC CCGATCGGCGAGAAACGTTTCCTGCCCCCTGAACCA CCCCCATCCTGGTCGGGCATCCGGAACGCCACACAC TTTCCCCCAGTGTGCCCCCAGAACATCCACACAGCT GTGCCCGAAGTCATGCTGCCAGTCTGGTTCACTGCC AACTTGGATATCGTCGCCACTTATATCCAGGAGCCCA ACGAAGATTGCCTCTATCTGAATGTGTATGTGCCCAC GGAAGATGGATCCGGCGCTAAGAAACAGGGCGAGGA CTTAGCGGATAATGACGGGGATGAAGATGAAGACATC

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CGAGACAGTGGTGCTAAACCTGTCATGGTCTACATCC ACGGAGGCTCTTACATGGAAGGAACAGGCAACATGA TTGATGGCAGTGTTCTTGCAAGTTACGGCAACGTCAT CGTCATCACCCTCAACTATCGGGTCGGGGTGCTAGG TTTCCTGAGCACTGGAGATCAGGCTGCCAAGGGCAA CTATGGGCTCCTTGATCAAATCCAGGCCCTTCGCTG GGTGAGTGAGAATATTGCCTTCTTTGGAGGAGATCC CCGTAGAATTACTGTCTTTGGCTCTGGCATCGGTGCA TCCTGTGTCAGTCTCCTTACACTGTCTCATCATTCTG AGGGGCTTTTCCAGAGGGCCATCATCCAAAGTGGCT CTGCTCTATCTAGCTGGGCTGTGAACTACCAACCAGT GAAGTATACCAGCTTGCTGGCAGACAAAGTGGGCTG TAACGTCCTGGACACTGTGGATATGGTGGATTGTCTT CGACAAAAGAGTGCCAAGGAGCTGGTAGAACAGGAC ATTCAGCCAGCCCGCTACCATGTGGCTTTTGGCCCT GTGATTGATGGTGATGTCATTCCTGATGACCCTGAGA TCCTTATGGAGCAGGGAGAGTTCCTCAACTATGATAT CATGCTAGGCGTCAACCAGGGTGAGGGTCTCAAGTT TGTGGAAGGGGTGGTGGACCCCGAGGATGGTGTCT CGGGCACTGACTTTGACTACTCTGTCTCCAATTTTG TGGACAATCTGTATGGCTATCCTGAGGGTAAGGACA CCCTGCGGGAGACTATCAAGTTCATGTATACGGACTG GGCAGACCGAGACAACCCTGAGACCCGCCGTAAAAC ACTGGTGGCACTCTTCACTGACCACCAGTGGGTGGA GCCTTCAGTGGTGACAGCCGATCTGCACGCCCGCTA TGGCTCACCTACCTACTTCTACGCCTTCTACCATCAC TGCCAGAGCCTCATGAAGCCCGCATGGTCAGATGCA GCACACGGGGATGAAGTGCCCTATGTTTTTGGTGTC CCTATGGTAGGTCCCACTGACCTTTTCCCCTGCAACT TCTCCAAGAATGATGTTATGCTCAGTGCTGTCGTCAT GACCTATTGGACCAACTTTGCCAAGACCGGGGATCC CAACAAGCCGGTACCCCAGGATACCAAGTTCATTCAC ACCAAGGCCAACCGCTTTGAGGAAGTGGCCTGGTCC AAATACAATCCCCGAGACCAGCTCTACCTTCACATC GGGCTGAAACCAAGGGTTCGTGATCATTACCGGGCC ACAAAGGTAGCCTTTTGGAAACACCTGGTGCCCCAC CTGTACAACCTGCATGACATGTTCCACTATACATCCA CGACCACCAAAGTGCCGCCCCCGGACACCACCCAC AGCTCCCACATCACCCGTAGGCCCAACGGCAAGACC TGGAGCACCAAGCGGCCGGCGATTTCACCTGCCTAC AGCAATGAGAATGCCCCTGGGTCCTGGAATGGGGAC CAGGATGCGGGGCCACTCCTGGTTGAGAACCCTCGA GACTACTCCACTGAATTAAGTGTCACTATCGCTGTGG GGGCCTCCCTCCTGTTTCTCAATGTGTTGGCCTTTG CTGCCCTCTATTACCGTAAGGACAAACGGCGCCAGG AGCCCCTGAGGCAGCCTAGCCCCCAAAGGGGAACTG GTGCCCCTGAATTGGGAACTGCTCCGGAGGAGGAGC TGGCAGCATTACAGTTGGGTCCCACTCACCATGAATG TGAGGCCGGTCCCCCACATGACACACTTCGCCTCAC

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AGCACTGCCCGACTATACCCTGACCCTGCGGCGCTC CCCTGATGACATCCCACTCATGACTCCCAACACCATC ACTATGATTCCTAATTCCCTGGTTGGGTTGCAGACCT TGCACCCCTATAACACCTTTGCCGCAGGGTTCAACA GTACTGGGCTGCCCCACTCACACTCCACTACCCGTG TATAA. 3. We use live cell imaging solution (Thermo Fisher, A14291DJ) for incubating cells with the fluorophore. Nevertheless, other medium (e.g., DMEM or RPMI) can also be used as they do not contain free cysteine that can react with the fluorophore. Serum should be avoided as the fluorophore seems to interact with the serum proteins. 4. We use RIPA buffer (Merck, R0278) with 1% of 100 protease inhibitor cocktail (Shanghai Epizyme Biomedical Technology, GRF101). 5. There are many commercial choices for preparing SDS-PAGE samples. We add 5% (v/v) β-mercaptoethanol into 4 LDS loading buffer (Thermo Fisher, NP0008). 6. Take about 0.5 mL of 0.01% poly-L-lysine solution to rinse each well in the 24-well plate (or 1 mL of 0.01% poly-lysine solution for 35 mm glass-bottom dishes), and then wash each well with PBS. Poly-lysine can enhance the attachment between the cells and culture plate. Thus, pre-treating the plate with poly-lysine can avoid cells from detaching from the plate in wash steps. However, a high level of poly-lysine is toxic to cells, so removal of residual poly-L-lysine by washing with PBS is required. 7. Complete culture medium with serum and antibiotics is freshly added to each well 30 min before transfection. 8. DTT is not stable in aqueous solution. Only use freshly prepared DTT solutions. 9. For a well in a 24-well plate, prepare the labeling mixture by adding 1 μL of 20 mM. FITC-PEG-TAMM and 0.2 μL of 2 M DTT into 198.8 μL of pre-warmed imaging buffer. The mixture is then added into the well.

Acknowledgements We thank the Shenzhen Bay Laboratory (SZBL) and National Natural Science Foundation of China (22277079) for financial support, as well as Mei Yu and Shixian Huang of the SZBL Bioimaging Core for assistance with ZEISS LSM980. We are grateful to Simon Elsa¨sser for sharing the sequence of pPylRS.

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References 1. Tamura T, Hamachi I (2019) Chemistry for covalent modification of endogenous/native proteins: from test tubes to complex biological systems. J Am Chem Soc 141(7):2782–2799. https://doi.org/10.1021/jacs.8b11747 2. Hoyt EA, Cal PMSD, Oliveira BL, Bernardes GJL (2019) Contemporary approaches to siteselective protein modification. Nat Rev Chem 3(3):147–171. https://doi.org/10.1038/ s41570-019-0079-1 3. Row RD, Prescher JA (2018) Constructing new bioorthogonal reagents and reactions. Acc Chem Res 51(5):1073–1081. https:// doi.org/10.1021/acs.accounts.7b00606 4. Haldon E, Nicasio MC, Perez PJ (2015) Copper-catalysed azide-alkyne cycloadditions (CuAAC): an update. Org Biomol Chem 13(37):9528–9550. https://doi.org/10. 1039/c5ob01457c 5. Li J, Kong H, Zhu C, Zhang Y (2020) Photocontrollable bioorthogonal chemistry for spatiotemporal control of bio-targets in living systems. Chem Sci 11(13):3390–3396. https:// doi.org/10.1039/c9sc06540g 6. Oliveira BL, Guo Z, Bernardes GJL (2017) Inverse electron demand Diels-Alder reactions in chemical biology. Chem Soc Rev 46(16): 4895–4950. https://doi.org/10.1039/ c7cs00184c 7. Zheng X, Li Z, Gao W, Meng X, Li X, Luk LYP, Zhao Y, Tsai YH, Wu C (2020) Condensation of 2-((Alkylthio)(aryl)methylene) malononitrile with 1,2-Aminothiol as a novel bioorthogonal reaction for site-specific protein modification and peptide cyclization. J Am Chem Soc 142(11):5097–5103. https://doi. org/10.1021/jacs.9b11875 8. Li X, Fekner T, Ottesen JJ, Chan MK (2009) A pyrrolysine analogue for site-specific protein ubiquitination. Angew Chem Int Ed Engl 48(48):9184–9187. https://doi.org/10. 1002/anie.200904472

9. Nodling AR, Spear LA, Williams TL, Luk LYP, Tsai YH (2019) Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells. Essays Biochem 63(2):237–266. https://doi.org/10.1042/ EBC20180042 10. de la Torre D, Chin JW (2021) Reprogramming the genetic code. Nat Rev Genet 22(3): 169–184. https://doi.org/10.1038/s41576020-00307-7 11. Zhou H, Cheung JW, Carpenter T, Jones SK Jr, Luong NH, Tran NC, Jacobs SE, Galbada Liyanage SA, Cropp TA, Yin J (2020) Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids. Bioorg Med Chem Lett 30(2): 126876. https://doi.org/10.1016/j.bmcl. 2019.126876 12. Watanabe R, Sato K, Hanaoka H, Harada T, Nakajima T, Kim I, Paik CH, Wu AM, Choyke PL, Kobayashi H (2014) Minibodyindocyanine green based activatable optical imaging probes: the role of short polyethylene glycol linkers. ACS Med Chem Lett 5(4): 4 1 1 – 4 1 5 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 1 / ml400533y 13. Sano K, Nakajima T, Miyazaki K, Ohuchi Y, Ikegami T, Choyke PL, Kobayashi H (2013) Short PEG-linkers improve the performance of targeted, activatable monoclonal antibodyindocyanine green optical imaging probes. Bioconjug Chem 24(5):811–816. https://doi. org/10.1021/bc400050k 14. Meineke B, Heimgartner J, Lafranchi L, Elsasser SJ (2018) Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells. ACS Chem Biol 13(11):3087–3096. https://doi.org/10. 1021/acschembio.8b00571

Chapter 15 Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Noncanonical Amino Acids Maria Kowalski-Jahn, Hannes Schihada, and Gunnar Schulte Abstract Here we describe the application of genetic code expansion and site-specific incorporation of noncanonical amino acids that serve as anchor points for fluorescent labeling to generate bioluminescence resonance energy transfer (BRET)-based conformational sensors. Using a receptor with an N-terminal NanoLuciferase (Nluc) and a fluorescently labeled noncanonical amino acid in the receptor’s extracellular part allows to analyze receptor complex formation, dissociation, and conformational rearrangements over time and in living cells. These BRET sensors can be used to investigate ligand-induced intramolecular (cysteine-rich domain [CRD] dynamics), but also intermolecular (dimer dynamics) receptor rearrangements. With the design of BRET conformational sensors based on the minimally invasive bioorthogonal labeling procedure, we describe a method that can be used in a microtiter plate format and can be easily adopted to investigate ligand-induced dynamics in various membrane receptors. Key words Conformational biosensors, Bioluminescence resonance energy transfer, G proteincoupled receptors, Amber codon suppression, Noncanonical amino acid, Bioorthogonal labeling, Molecular dynamics, Microtiter plate reader

1

Introduction Agonist-induced activation of G protein-coupled receptors (GPCRs) is accompanied by structural dynamics as a consequence of ligand-receptor interaction, receptor activation, potential receptor dimer dynamics, and receptor coupling to intracellular transducer proteins. While detection of distinct receptor activity states (inactive vs active, for example) has been possible using biochemical methodology, the advent and further development of bioluminescence resonance energy transfer (BRET) provides exciting opportunities to analyze receptor complex formation, dissociation, and conformational rearrangements over time and in living cells [1– 4]. BRET relies on energy transfer between a bioluminescent donor and a fluorescent acceptor that are in proximity of less than 100 A˚. While the BRET efficiency depends on the distance between BRET

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_15, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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donor and BRET acceptor, also their relative orientation has an important impact on the efficiency of resonance energy transfer [5, 6]. Thus, recorded BRET values need to be interpreted as a composite measure of distance and orientation of the donor to the acceptor. In the field of receptor pharmacology, BRET is routinely used to quantify protein-protein interaction, such as GPCR agonist-induced G protein dissociation, GPCR-arrestin interaction, or similar [7–10]. These readouts monitor BRET between two distinct proteins (intermolecular) but also intramolecular BRET designs can be used to study rearrangements within a single protein. This technique is often used in artificial BRET constructs, such as second messenger-detecting biosensors for cAMP or DAG [11– 13], but also structural rearrangements as a consequence of, for example, GPCR activation can be visualized as ΔBRET in so-called conformational sensors [14–17]. Furthermore, BRET-based ligand binding assays are available, and the way BRET can be used to quantify pharmacological responses is only limited by the inventor’s fantasy. An obvious caveat of the methodology is, however, the requirement for tagging the proteins of interest with relatively large tags, such as a luciferase as an energy donor and a fluorescent protein as acceptor. The larger the introduced tag, the more severe can the consequences for the protein of interest be. Thus, many attempts have been made over the years to reduce the size of the respective moieties, which has resulted in the creation of NanoLuciferase (NanoLuc, Nluc) or fluorescein arsenic hairpin binder (FlAsH)-binding motifs [18–20]. Here, we report another valuable, minimally invasive labeling procedure based on genetic code expansion and site-specific incorporation of noncanonical amino acids serving as anchors for fluorescent labelling via a subsequent bioorthogonal coupling reaction (strain-promoted inverse electron-demand Diels-Alder cycloaddition [SPIEDAC]). While labeling of receptors with this approach was reported before to visualize membrane-embedded proteins [21, 22] and to investigate the molecular mechanism of adhesion GPCR activation by means of fluorescence resonance energy transfer (FRET) efficiency quantification [23], we were the first to combine orthogonal receptor labeling with the detection of ligand-induced BRET changes over time [16]. We have used this minimally invasive technique to investigate agonist-induced rearrangements in class F (frizzled; FZD) GPCRs. Introduction of an N-terminal Nluc as BRET donor and a small-molecule fluorescent moiety in the extracellular loops of the transmembrane core of the receptor or the linker domain as BRET acceptor allowed us to pinpoint structural dynamics in response to agonist exposure (Fig. 1). The broad applicability of the approach is underlined by its ability to report on intramolecular (FZD-cysteine-rich domain [CRD] dynamics—where BRET donor and acceptor are in the same protein) and intermolecular (FZD-dimer dynamics—with BRET donor and acceptor on

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Fig. 1 Workflow of BRET-based GPCR extracellular conformational biosensors. The BRET sensors are based on bioorthogonal labeling of noncanonical amino acids and are generated in a multi-step work procedure with (1) a structure-guided sensor design, (2) generation of sensor variants, (3) validation of the sensor variants, and (4) BRET experiments. Due to the unpredictable change in relative orientation of the energy partners, ligand stimulation can cause a decrease or increase in BRET. Tet-Cy3, tetrazine-Cy3, TCO*K, trans-cyclooct2-ene-L-lysine

different proteins) protein dynamics (Fig. 2). Thus, combining genetic code expansion and orthogonal labeling with BRET allowed for the first time to monitor how WNT proteins induce a conformational rearrangement in the extracellular domains of FZDs.

2

Materials All solutions should be prepared using ultrapure water and analytical-grade reagents.

2.1 Cloning of Amber Codon-Bearing Receptor (FZD6) Constructs

1. FZD6 plasmid DNA with a C-terminal tag (e.g., 1D4). 2. GeneArt Site-Directed Mutagenesis System (Thermo Fisher Scientific) for cloning the amber mutants (see Note 1). 3. Subcloning of amber codon-bearing FZD and NanoLuciferase (Nluc) tag into an expression plasmid carrying four repeats of the orthogonal suppressor tRNA (Addgene number: 140008) using the NEBuilder HiFi DNA Assembly kit (New England Biolabs). Alternatively, distinct target proteins bearing either the amber codon or extracellular Nluc can be combined to assess intermolecular BRET. 4. Primers. We design the corresponding Amb mutant primers using the GeneArt Primer and Construct Design Tool (Thermo Fisher). We use NEBuilder to design primers for subcloning amber codon-bearing FZD and NanoLuciferase tag into the tRNA expression vector.

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Fig. 2 Intra- vs. intermolecular BRET responses. Schematic depiction of intramolecular (BRET donor and acceptor in the same protein) and intermolecular (BRET donor and acceptor on different proteins) protein dynamics to analyze extracellular conformational rearrangements within the protein or receptor dimerization dynamics

2.2

Cell Culture

1. Human embryonic kidney (HEK)-293 cells, HEK-293A or HEK-293T (ATCC) cells (see Note 2). 2. Cell culture medium: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (100 U/mL penicillin; 0.1 mg/mL streptomycin). 3. Dulbecco’s phosphate-buffered saline (DPBS, without CaCl2 and MgCl2). 4. T75 cell culture flasks. 5. Single-channel and multichannel pipettes.

2.3 Counting and Plating

1. 0.05% trypsin, 0.53 mM EDTA in ddH2O. 2. Hemocytometer. 3. Trypan blue.

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4. Colorless-wall, black-wall and black-bottomed, white-wall and white-bottomed 96-well microtiter plates, 24-well plates (see Note 3). 5. 0.1 mg/mL poly-D-Lysine (PDL) in ddH2O. 2.4 Amber Codon Suppression

1. Noncanonical amino acid (TCO*K, Sichem, SC-8008).

trans-cyclooct-2-ene-L-lysine

2. 0.2 M NaOH, 15% DMSO for preparation of a 100 mM TCO*K stock solution. 2.5

Transfection

1. Nluc-FZD6-amber codon plasmid DNA and pcDNA3.1 plasmid DNA. 2. ftRNA/tRNA synthetase plasmid DNA (Addgene number: 140023). 3. Lipofectamine 2000 (Thermo Fisher). 4. 1.5 mL or 2 mL microcentrifuge tubes, or 15 mL conical tubes. 5. Opti-MEM.

2.6

Immunoblotting

1. 2
Laemmli buffer: 125 mM Tris–HCl (pH 6.8), 20% glycerol, 4% SDS, 0.05% bromophenol blue, 200 mM DTT. 2. Sonicator. 3. 7.5% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). 4. Trans-Blot Turbo Transfer System (Bio-Rad). 5. Trans-Blot Turbo RTA Midi 0.45 μm LF PVDF Transfer Kit (Bio-Rad). 6. TBS-T: 25 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.6. 7. 5% (w/v) low-fat milk powder in TBS-T. 8. Primary antibodies (e.g., anti-Nluc, R&D Systems, MAB100261-SP, mouse, 1 μg/mL in 5% milk/TBS-T; anti1D4, National Cell Culture Center, mouse, 10 μg/mL in 5% milk/TBS-T). 9. Secondary antibody conjugated to horseradish peroxidase (e.g., goat anti-mouse IgG, Thermo Fisher Scientific, 31430, 0.16 μg/mL in TBS-T). 10. Western ECL Substrate.

2.7 Surface Expression

1. 1
DPBS (supplemented with CaCl2 and MgCl2). 2. ELISA buffer: 1% bovine serum albumin (BSA) in 1
DPBS. 3. Wash buffer: 0.5% bovine serum albumin (BSA) in 1
DPBS. 4. Primary antibody (anti-Nluc antibody). 5. Horseradish peroxidase–conjugated secondary antibody.

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6. 3,30 ,5,50 -Tetramethylbenzidine (TMB) liquid substrate system (cat. T8665 from Merck). 7. 2 M hydrochloric acid (HCl). 8. Microtiter plate reader. 2.8 Fluorescence Labeling

1. 10 mM tetrazine-Cy3 (Tet-Cy3, Jena Bioscience, CLK-01405) in DMF. 2. Hank’s Balanced Salt Solution (HBSS). 3. 0.1% BSA in HBSS. 4. Microtiter plate reader equipped with filters for Cy3 (Ex. 544 nm/Em. 590 nm).

2.9 BRET Measurement

1. Furimazine (NanoBRET™ Nano-Glo® Substrate, Promega). 2. Microtiter plate reader CLARIOstar equipped with monochromators to separate Nluc (450/80 nm) and Cy3 (580/30 nm) emission. 3. FZD ligands (e.g., WNT-3A, WNT-5A, R&D systems/BioTechne).

2.10

Data Analysis

1. Western blot developing control and analysis software. 2. Microtiter plate reader control software and analysis software. 3. Spreadsheet application (e.g., MS Excel). 4. Statistical software (e.g., GraphPad Prism).

3

Methods

3.1 Cloning of FZD Constructs

For a maximal amber suppression and fluorescent labeling efficiency, a structure-based selection of amber codon sites is recommended. Therefore, the relative location within the amino acid chain (better N-terminal than C-terminal for the sake of receptor expression), the amino acid’s orientation (favor amino acids pointing outwards to allow accessibility for the fluorescent dye), and finally the distance between N-terminal Nluc tag and labeling site should be taken into consideration. 1. Introduce the amber codon (TAG) into the receptor by using site-directed mutagenesis. 2. Subclone the NanoLuciferase (Nluc) tag at the N terminus of the receptor, e.g., using a DNA assembly kit. 3. To boost the amber codon suppression, introduce the respective Nluc-tagged amber codon-bearing receptor into an expression plasmid carrying four repeats of the orthogonal suppressor pyrrolysyl-tRNA (Addgene number: 140008) by using a DNA assembly kit (Fig. 1).

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3.2 Cell Culture, Counting, Seeding, and Transfection

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1. Precoat 96-well microtiter plates with 50 μL/well of a 0.1 mg/ mL poly-D-lysine (PDL) solution and incubate at room temperature for 30 min. 2. Aspirate the coating solution and wash twice with 150 μL/well of DPBS. 3. Let the plate dry for about 2 h in the laminar flow hood. 4. Grow HEK-293T cells in cell culture medium at 37  C and 5% CO2. 5. Passage cells when they reach confluency of 80%. 6. Aspirate medium and wash cells carefully with 10 mL of DPBS. 7. Incubate cells with 1 mL of trypsin-EDTA solution for 5 min at 37  C and 5% CO2 and resuspend in 9 mL of fresh cell culture medium. 8. Transfer 1 mL of the resuspended cells to a flask with fresh cell culture medium. 9. To determine the cell density, mix 10 μL of the cell suspension with 10 μL of trypan blue and transfer 10 μL of the mixed suspension to a hemocytometer. 10. Determine the number of unstained, living cells in one large square and calculate the cell number/mL suspension according to the following equation: Cell number ¼ counted cells  2ðdilution factorÞ  104 mL 11. Dilute the cell suspension to obtain a density of 25,000 cells/ mL for a 96-well microtiter plate and 100,000 cells/mL for a 24-well plate and seed 100 μL of cell suspension in each well of a 96-well microtiter plate or 500 μL of the cell suspension in each well of a 24-well plate, respectively. 12. Twenty-four hours after seeding, HEK-293T cells in a 24-well plate were transiently transfected with a defined transfection ratio of 9:1 (see Note 4) with 0.45 μg (96-well microtiter plate: 0.09 μg) of the indicated receptor constructs and 0.05 μg (96-well microtiter plate: 0.01 μg) of pyrrolysyl-tRNA/tRNA synthetase plasmid per well. Control conditions were balanced with pcDNA3.1. 13. For transfection in a 24-well plate, add the respective amount of amber codon-bearing receptor construct and pyrrolysyltRNA/tRNA synthetase to 25 μL of Opti-MEM in a microcentrifuge tube and incubate for 5 min at room temperature. 14. Add 1 μL of Lipofectamine 2000 to 25 μL Opti-MEM in a separate microcentrifuge tube and let incubate for 5 min at room temperature.

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15. Add the Lipofectamine 2000/Opti-MEM mix to the plasmid DNA/Opti-MEM mix, mix the tube, and incubate for 10 min at room temperature. 16. For transfection in a 96-well microtiter plate, use 15 μL of Opti-MEM (instead of 25 μL in the 24-well format) for the plasmid DNA/Opti-MEM mix and additional 15 μL of OptiMEM for the Lipofectamine 2000/Opti-MEM mix. 17. Meanwhile, remove the cell culture medium from cells and add 450 μL (24-well plate) or 70 μL (96-well microtiter plate) of fresh medium substituted with a final concentration of 0.1 mM trans-cyclooct-2-ene-L-lysine (TCO*K) or vehicle to each well. 18. Subsequently, add 50 μL (24-well plate) or 30 μL (96-well microtiter plate) of the plasmid DNA/Lipofectamine 2000 solution in Opti-MEM into each well. 19. Mix gently the plate and culture at 37  C and 5% CO2 for 48 h. 3.3 Immunoblotting to Assess for Amber Codon Suppression

1. Seed 100,000 HEK-293T cells/well in 24-well plates the day prior to transfection. 2. Transfect the cells with a defined transfection ratio of 9:1 with 0.45 μg of the amber codon-bearing receptor constructs and 0.05 μg of pyrrolysyl-tRNA/tRNA synthetase (control conditions are balanced with pcDNA3.1) per well as described in Subheading 3.2 in detail (see Note 5). 3. Add the noncanonical amino acid TCO*K or vehicle to a final concentration of 0.1 mM into each well and let the cells culture at 37  C and 5% CO2 for 48 h. 4. After 48 h, lyse cells in 2
Laemmli buffer, transfer the lysed cells into microcentrifuge tubes, and sonicate the samples. 5. Place microcentrifuge tubes before and after sonication on ice. 6. Load 15 μL of each sample on a 7.5% gel and run the SDS-PAGE according to the supplier’s information. 7. Transfer the separated proteins to a polyvinylidene difluoride membrane with the Trans-Blot® Turbo Transfer System (see Note 6). 8. After transfer, incubate the membrane in 5% low-fat milk/ TBS-T and subsequently in primary antibodies overnight at 4  C for detection of a C-terminal tag to ensure a proper amber codon suppression (e.g., anti-1D4 antibody). 9. On the next day, wash the membranes four times in TBS-T, and incubate with respective secondary antibodies conjugated to horseradish peroxidase. 10. Wash membranes again four times with TBS-T. 11. Develop the membranes using Western ECL Substrate according to the supplier’s information.

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3.4 Whole-Cell ELISA to Confirm Receptor Surface Expression

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1. For quantification of cell surface receptor expression, plate 15,000 HEK-293T cells/well in colorless PDL-precoated 96-well microtiter plates. 2. On the next day, transfect cells with 0.09 μg of the amber codon-bearing receptor constructs including an N-terminal Nluc tag and 0.01 μg of pyrrolysyl-tRNA/tRNA synthetase. 3. Culture the cells in the absence or presence of a final concentration of 0.1 mM TCO*K at 37  C and 5% CO2. 4. After 48 h, remove the medium from the cells and incubate the cells with 50 μL of an anti-Nluc antibody (e.g., R&D systems, MAB100261-SP, mouse, 1:500) in 1% BSA/DPBS for 1 h at 4  C to detect the Nluc tag fused to the N terminus of the receptor. 5. Following incubation, wash the cells five times with 100 μL of 0.5% BSA/DPBS each for 5 min and remove the wash solution by tapping the plate on a paper towel. 6. After the last washing step, add 50 μL of a horseradish peroxidase–conjugated secondary antibody at an appropriate dilution in 1% BSA/DPBS for 1 h at 4  C. 7. Wash the cells again five times with 100 μL of 0.5% BSA/DPBS each for 5 min and remove the wash solution by tapping the plate on a paper towel. 8. Add 50 μL of the peroxidase substrate TMB and incubate for 30 min light-protected at room temperature. 9. To stop the reaction by acidification, add 50 μL of 2 M HCl, mix gently the plate, and read the absorbance at 450 nm using a microtiter plate reader.

3.5 Assessment of Fluorescence Labeling Efficiency

1. For quantification of the fluorescence labeling of the receptor mutants, plate 15,000 HEK-293T cells in black PDL-precoated 96-well microtiter plates. 2. On the next day, transfect cells with 0.09 μg of the amber codon-bearing receptor constructs and 0.01 μg of tRNA/ tRNA synthetase and culture the cells in the absence or presence of 0.1 mM TCO*K at 37  C and 5% CO2. 3. Forty-eight hours after transfection, wash cells in DPBS and keep them for 2 h in full cell culture medium at 37  C and 5% CO2. 4. Subsequently, label receptor-expressing cells with 1 μM Tet-Cy3 for 30 min at 37  C and 5% CO2. 5. Wash cells again with DPBS and keep them for additional 30 min in full cell culture medium at 37  C and 5% CO2. 6. Repeat the washing step with DPBS. 7. Exchange DPBS with 0.1% BSA/HBSS and read fluorescence intensities using a microtiter plate reader equipped with filters for Cy3 (Ex. 544 nm/Em. 590 nm) (see Note 7).

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3.6 Detecting Ligand-Induced Conformational Changes in Nluc-FZD with BRET

1. For inter- and intramolecular Nluc-FZD BRET measurements, plate 15,000 HEK-293T cells in white PDL-precoated 96-well microtiter plates. 2. On the next day, transfect cells with 0.09 μg of the amber codon-bearing receptor construct and an N-terminal Nluc tag on the same or two different proteins of interest and 0.01 μg of tRNA/tRNA synthetase. Culture cells in the presence of 0.1 mM TCO*K at 37  C and 5% CO2. 3. Forty-eight hours after transfection, wash cells in DPBS and keep them for 2 h in DMEM at 37  C and 5% CO2. 4. Subsequently, label receptor-expressing cells with 1 μM Tet-Cy3 for 30 min at 37  C and 5% CO2. 5. Wash cells again with DPBS and keep them for an additional 30 min in DMEM at 37  C and 5% CO2. 6. Next, wash cells with DPBS, exchange DPBS with 90 μL of a 1/1000 dilution of furimazine stock solution (Promega) (see Note 8) in 0.1% BSA/HBSS and incubate for 5 min. 7. Measure basal BRET in three consecutive reads using a CLARIOstar microtiter plate reader (BMG Labtech, Ortenberg, Germany) (see Note 9) equipped with monochromators to separate Nluc (450/80 nm) and Cy3 (580/30 nm) emission. 8. After the first three reads, add 10 μL of at least one concentration (3 μg/mL, or increasing) of WNT-3A or WNT-5A solution (in 0.1% BSA/HBSS) or vehicle control to each well and record the ratio for an additional 25–60 min. 9. For kinetic experiments with a higher temporal resolution, record eight baseline BRET reads within 2 min prior to manual addition of compounds or vehicle control, followed by at least 40 reads.

3.7

Data Analysis

1. Open the BRET and fluorescence data in Excel or a similar spreadsheet application. 2. Calculate the raw BRET ratio for each well at each individual time point according to the following equation: Raw BRET ratio ¼

Emission intensity in the BRET acceptor channel Emission intensity in the BRET donor channel

3. To quantify ligand-induced BRET changes, use the raw BRET values before (BRETbasal) and after (BRETstim) ligand/vehicle application for each well to calculate the raw ΔBRET according to the following equation: Raw ΔBRET ½% ¼ 100 

ðBRETstim  BRETbasal Þ BRETbasal

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4. Finally, to correct for nonspecific, vehicle-induced BRET signals, the ΔBRET values can be calculated by subtraction of the average raw ΔBRET of the vehicle control from the raw ΔBRET of each ligand-treated well according to the following equation: ΔBRET ½% ¼ raw ΔBRETligand  average raw ΔBRETvehicle

4

Notes 1. Use the GeneArt Site-directed mutagenesis system or any other method for site-directed mutagenesis. 2. All specifications apply to HEK-293T cells. Other, transfectable cell lines can also be used; optimization of cell culture, transfection techniques, and plating density may be required. 3. For bottom reading in a microtiter plate reader, use a transparent-bottom white- or black-wall 96-well plate. 4. In our hands, the transfection ratio was optimized for class F receptors by transfecting different ratios of receptor construct and tRNA/tRNA synthetase and subsequently analyzing the receptor total expression by SDS-PAGE/Western blot. For different receptors the transfection ratio may be optimized. 5. For analyzing amber codon suppression of the mutated receptors in comparison to wild type on the same Western blot, it might be necessary to dilute the wild-type receptor with pcDNA for transient transfection of HEK-293T cells. 6. For analyzing the total receptor expression, we have used the SDS-PAGE/transfer kit/detection system from Bio-Rad. Any other SDS-PAGE/Western blot supplies may be used. 7. In addition to measuring the fluorescence labeling efficiency in a microtiter plate reader, HEK-293T cells can also be seeded in glass-bottom PDL-precoated 96-well microtiter plates and fluorescence of the labeled cells can be assessed by fluorescence microscopy with filters for Cy3 (Ex. 544 nm/Em. 590 nm). 8. In our hands, the furimazine preparation provided in the NanoBRET™ Nano-Glo® Substrate kit from Promega (#N157A) yielded high assay sensitivity when applied in a 1: 1000 dilution in 0.1% BSA/HBSS. Higher dilutions may be tested for their effect on the dynamic range of the BRET conformational sensors. 9. Our assays were optimized using the CLARIOstar microtiter plate reader from BMG Labtech with a 450/80 nm and 580/30 nm filter set. Any other plate reader with luminescence and fluorescence detection system can also be used.

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Acknowledgments Most importantly, we thank Simon J. Elsa¨sser, Birthe Meineke and Thomas P. Sakmar for advice and tools to set up the orthogonal labeling of FZDs. We also thank Anna Krook for access to the CLARIOstar microtiter plate reader. The work was supported by grants from Karolinska Institutet, the Swedish Research Council (2019-01190), the Swedish Cancer Society (CAN2017/561, 20 1102PjF), the Novo Nordisk Foundation (NNF20OC0063168, NNF19OC0056122), and the German Research Foundation (DFG; KO 5463/1-1, 427840891). References 1. Lohse MJ, Nuber S, Hoffmann C (2012) Fluorescence/bioluminescence resonance energy transfer techniques to study g-protein-coupled receptor activation and signaling. Pharmacol Rev 64:299–336 2. Salahpour A, Espinoza S, Masri B et al (2012) BRET biosensors to study GPCR biology, pharmacology, and signal transduction. Front Endocrinol (Lausanne) 3:105 3. Picard LP, Scho¨negge AM, Lohse MJ, Bouvier M (2018) Bioluminescence resonance energy transfer-based biosensors allow monitoring of ligand- and transducer-mediated GPCR conformational changes. Commun Biol 1:106. https://doi.org/10.1038/s42003-0180101-z 4. Schihada H, Vandenabeele S, Zabel U et al (2018) A universal bioluminescence resonance energy transfer sensor design enables highsensitivity screening of GPCR activation dynamics. Commun Biol 1:105. https://doi. org/10.1038/s42003-018-0072-0 5. Jensen JB, Lyssand JS, Hague C, Hille B (2009) Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K + channels. J Gen Physiol 133:347–359. https:// doi.org/10.1085/jgp.200810075 6. Ziegler N, Ba¨tz J, Zabel U et al (2011) FRETbased sensors for the human M1-, M3-, and M 5-acetylcholine receptors. Bioorganic Med Chem 19:1048–1054. https://doi.org/10. 1016/j.bmc.2010.07.060 7. Pfleger KDG, Dalrymple MB, Dromey JR, Eidne KA (2007) Monitoring interactions between G-protein-coupled receptors and β-arrestins. In: Biochemical society transactions. Portland Press Ltd, pp 764–766 8. Donthamsetti P, Quejada JR, Javitch JA et al (2015) Using bioluminescence resonance

energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors. Curr Protoc Pharmacol 70: 2.14.1–2.14.14. https://doi.org/10.1002/ 0471141755.ph0214s70 9. Schihada H, Shekhani R, Schulte G (2021) Quantitative assessment of constitutive G protein–coupled receptor activity with BRETbased G protein biosensors. Sci Signal 14: e a b f 1 65 3 . h t t p s : //d o i . or g / 1 0 . 1 1 2 6 / scisignal.abf1653 10. Masuho I, Martemyanov KA, Lambert NA (2015) Monitoring G protein activation in cells with BRET. Methods Mol Biol 1335: 107–113. https://doi.org/10.1007/978-14939-2914-6_8 11. Wright SC, Bouvier M (2021) Illuminating the complexity of GPCR pathway selectivity – advances in biosensor development. Curr Opin Struct Biol 69:142–149 12. Namkung Y, LeGouill C, Kumar S et al (2018) Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors. Sci Signal 11: eaat1631. https://doi.org/10.1126/scisignal. aat1631 13. Barak LS, Salahpour A, Zhang X et al (2008) Pharmacological characterization of membrane-expressed human trace amineassociated receptor 1 (TAAR1) by a bioluminescence resonance energy transfer cAMP biosensor. Mol Pharmacol 74:585–594. https:// doi.org/10.1124/mol.108.048884 14. Sleno R, Pe´trin D, Devost D et al (2016) Designing BRET-based conformational biosensors for G protein-coupled receptors. Methods 92:11–18. https://doi.org/10.1016/j. ymeth.2015.05.003

Genetically Encoded BRET Sensors 15. Schihada H, Schihada H, Ma X et al (2020) Development of a conformational histamine H3 receptor biosensor for the synchronous screening of agonists and inverse agonists. ACS Sensors 5:1734–1742. https://doi.org/ 10.1021/acssensors.0c00397 16. Kowalski-Jahn M, Schihada H, Turku A et al (2021) Frizzled BRET sensors based on bioorthogonal labeling of non-canonical amino acids reveal WNT-induced dynamics of the cysteine-rich domain. Sci Adv 7:eabj7917. https://doi.org/10.1126/sciadv.abj7917 17. Schihada H, Nemec K, Lohse MJ, Maiellaro I (2021) Bioluminescence in G protein-coupled receptors drug screening using nanoluciferase and halo-tag technology. In: Methods in molecular biology. Humana Press Inc, pp 137–147 18. Hall MP, Unch J, Binkowski BF et al (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848. https:// doi.org/10.1021/cb3002478 19. Adams SR, Campbell RE, Gross LA et al (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and

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Chapter 16 Selective Inhibition of Kinase Activity in Mammalian Cells by Bioorthogonal Ligand Tethering Jinghao Chen, Yang Huang, Wen-Biao Gan, and Yu-Hsuan Tsai Abstract Enzymes are critical for cellular functions, and malfunction of enzymes is closely related to many human diseases. Inhibition studies can help in deciphering the physiological roles of enzymes and guide conventional drug development programs. In particular, chemogenetic approaches enabling rapid and selective inhibition of enzymes in mammalian cells have unique advantages. Here, we describe the procedure for rapid and selective inhibition of a kinase in mammalian cells by bioorthogonal ligand tethering (iBOLT). Briefly, a non-canonical amino acid bearing a bioorthogonal group is genetically incorporated into the target kinase by genetic code expansion. The sensitized kinase can react with a conjugate containing a complementary biorthogonal group linked with a known inhibitory ligand. As a result, tethering of the conjugate to the target kinase allows selective inhibition of protein function. Here, we demonstrate this method by using cAMP-dependent protein kinase catalytic subunit alpha (PKA-Cα) as the model enzyme. The method should be applicable to other kinases, enabling their rapid and selective inhibition. Key words Kinase, Protein phosphorylation, Bicyclo[6.1.0]nonyne-lysine, Genetic code expansion, Biorthogonal labeling

1

Introduction Enzymes are proteins that catalyze reactions in biosystems, and malfunction of enzymes is commonly associated with many human diseases [1–3]. Thus, enzymes have been the focus of many research areas. The physiological roles of enzymes are generally elucidated through inhibition studies. Ideally, enzyme inhibition must be specific, to avoid unwanted side effects, and fast, to provide the time resolution and prevent adaptive compensation by the cell. This is particularly important when validating enzymes as therapeutic targets. However, for many enzymes, selective and rapid inhibition in cells is still an outstanding challenge [4]. Genetic approaches (e.g., gene knockdown and knockout) can offer unprecedented specificity to target any enzymes in the cell [5]. However, the lag time is long, which often leads cells to

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_16, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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establish compensatory mechanisms that mask the biology of the system under investigation [6]. In contrast, chemical inhibition by small molecules is fast [5], but highly specific small-molecule modulators are only available for a minor fraction of the known enzymes [7]. Chemogenetic inhibition combines the advantages of both genetic (i.e., specific) and chemical (i.e., rapid) approaches [4, 8]. Here, a genetic modification is introduced into the enzyme of interest, a process known as “sensitization,” so that the target enzyme becomes sensitive to a designer small molecule that otherwise has no affinity to the wild-type enzyme or other family members. We have developed a chemogenetic approach based on bioorthogonal tethering (Fig. 1) [9, 10]. In this approach, a sensitized target is generated by placing a noncanonical amino acid bearing a bioorthogonal group into the target enzyme through genetic code expansion [11]. A known inhibitor is repurposed to generate a conjugate with the complementary bioorthogonal group. Thus, the sensitized target can be selectively inhibited by the inhibitor conjugate due to covalent tethering of the conjugate through the biorthogonal groups. Using this approach, we demonstrated selective and rapid inhibition of intracellular kinases MEK1, MEK2, and LCK, for which no selective small-molecule inhibitors exist [9]. In this chapter, we describe a step-by-step procedure for selective inhibition by bioorthogonal ligand tethering (iBOLT) in HEK293T cells. Here, cAMP-dependent protein kinase catalytic subunit alpha (PKA-Cα) is used as the model enzyme and bicyclo [6.1.0]nonyne lysine (BCNK) as the noncanonical amino acid bearing a bioorthogonal functionality. From the reported crystal structure of PKA-Cα and an inhibitor [12], we select three residues, G53, Q85, and K193, for incorporation of BCNK (Fig. 2). Based on the crystal structure, small-molecule conjugate Tet-PKA for bioorthogonal tethering is designed, where a linker connecting the complementary bioorthogonal group, tetrazine, is attached to the binding inhibitor (Fig. 3). For incorporation of BCNK into PKA-Cα, an amber codon is used to replace the codon of the corresponding amino acid residue. Cells are transfected with a plasmid encoding an evolved orthogonal aminoacyl-tRNA synthetase/tRNA pair, a plasmid encoding PKA-Cα with an amber codon, and a reporter plasmid. For detecting PKA activity by immunoblotting, a green fluorescent protein (GFP) with a C-terminal PKA recognition sequence LRRATLVD [13] is used as the reporter. For tracking real-time PKA activity by confocal microscopy, a ratiometric reporter is employed [14].

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Fig. 1 Concept of selective inhibition by bioorthogonal ligand tethering (iBOLT). Noncanonical amino acid BCNK ① is fed to HEK293T cells expressing an orthogonal aminoacyl-tRNA synthetase/tRNA pair and the target gene with a TAG codon for its incorporation. The sensitized protein bearing BCNK could react with the small-molecule conjugate ② via the biorthogonal reaction ③. Upon reaction, the inhibitory group on the conjugate bind to the sensitized protein and inhibit its function

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Materials All commercial chemicals are of reagent grade or higher. All solution is prepared using ultrapure water or PBS solution at common concentration.

2.1 Small-Molecule Conjugate Tet-PKA

1. N-BOC-6-aminocaproic acid. 2. (R)-3-(Boc-amino)piperidine. 3. 4-Cyanobenzoic acid. 4. 4,5-Dichloro-7H-pyrrolo[2,3-d]pyrimidine. 5. HATU (CAS 148893-10-1).

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Fig. 2 Structure of PKA-Cα with inhibitor (R)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-3-amine (PDB: 3MVJ). PKA-Cα and the inhibitor/ligand are shown in grey and orange, respectively. The mutation sites, G53, Q85, and K193, are highlighted in blue

Fig. 3 Synthetic scheme of Tet-PKA. Reagents and conditions: (a) NaHCO3, EtOH, reflux, 77%; (b) HCl, dioxane, 95%; (c) N-Boc-6-aminocaproic acid, EDC, DIPEA, DMF, 43%; (d) HCl, dioxane, 97%; (e) hydrazine monohydrate, CH2Cl2, EtOH, 50 °C; NaNO2, AcOH, 55%; (f) A5, HATU, DIPEA, THF, 62%

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6. 4 N HCl in dioxane. 7. Hydrazine monohydrate. 8. Sulfur. 9. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). 10. N,N-Diisopropylethylamine (DIPEA). 11. Sodium bicarbonate. 12. Sodium nitrite. 13. Anhydrous dimethylformamide (DMF). 14. Anhydrous tetrahydrofuran (THF). 15. Acetic acid. 16. Dichloromethane. 17. Ethanol. 18. Ethyl acetate. 19. Methanol. 20. Petroleum ether. 21. Water. 22. Microwave reaction tube and microwave reactor. 23. 25 mL round-bottom flasks. 24. Magnetic stirrer with heating function. 25. Reflux condenser. 26. Oil bath. 27. Separating funnels. 28. Brine. 29. Anhydrous Na2SO4. 30. Chromatography columns. 31. NMR spectrometer. 32. Mass spectrometer. 33. 1 mM Tet-PKA: Dissolve 5 mg of Tet-PKA in 910 μL of DMSO to make a solution of 10 mM. Prepare 1 mM stock solution by diluting the 10 mM solution with DMSO (see Note 1). 2.2

Plasmids

1. pPylRS(AF) [15]: This plasmid, available on Addgene (#140023), encodes an evolved PylRS under the EF-1α promoter and four copies of Pyl tRNACUA under the human 7SK promoter, enabling site-specific incorporation of BCNK in response to the amber codon. 2. pPKA-Cα: This plasmid encodes the model kinase protein, a constitutively active cAMP-dependent protein kinase catalytic subunit alpha (PKA-Cα; see Note 2) and four copies of Pyl tRNACUA by replacing the evolved PylRS in pPylRS(AF) with

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PKA-Cα through NcoI and SalI sites. For BCNK incorporation, six bases before and after the amber codon are optimized using iPASS (www.bultmannlab.eu/tools/iPASS) [16], and the production of Pyl tRNACUA from both this plasmid and pPylRS(AF) should enhance the incorporation efficiency of BCNK [17, 18]. 3. pGFP-LRRATLVD: This plasmid expressing a green fluorescent protein (GFP) with a C-terminal PKA substrate sequence (LRRATLVD) is used to detect PKA inhibition in cells. The Thr residue of LRRATLVD is specifically phosphorylated by PKA in HEK293T cells, which can be recognized by a specific antibody and monitored by immunoblotting. 4. pExRai-AKAR2 [14]: This plasmid, available on Addgene (#161753), is used as the reporter in high-content live cell imaging. 2.3 Reagents and Consumables

1. Fetal bovine serum (FBS).

2.3.1 Cell Culture and Transfection

3. 10,000 U/mL penicillin-streptomycin (P/S).

2. DMEM. 4. Growth media: Add 50 mL of FBS and 5 mL of P/S into 445 mL of DMEM. 5. 0.25% trypsin. 6. Transfection reagent (e.g., Lipofectamine 2000). 7. Opti-MEM. 8. PBS solution. 9. 0.1% poly-lysine solution. 10. 100 mM BCNK: Dissolve 32 mg bicyclo[6.1.0]nonyne-Lysine (BCNK) in 1 mL of 0.1 M NaOH (see Note 3).

2.3.2 Cell Lysis and SDSPAGE

1. RIPA buffer (see Note 4). 2. Protease inhibitor cocktail. 3. Phosphatase inhibitor cocktail. 4. β-Mercaptoethanol. 5. SDS-PAGE sample loading buffer (see Note 5). 6. 12.5% SDS-PAGE preparation kit. 7. Tris-glycine-SDS running buffer.

2.3.3

Immunoblotting

1. 0.45 μm nitrocellulose membranes. 2. 1 M Tris–HCl (pH 10.5). 3. Anode buffer 1: Mix 300 mL of 1 M Tris (pH 10.5), 100 mL of MeOH, and 600 mL of ddH2O. 4. Anode buffer 2: Mix 25 mL of 1 M Tris (pH 10.5), 100 mL of MeOH, and 875 mL of ddH2O.

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5. Cathode buffer: Mix 25 mL of 1 M Tris (pH 10.5), 100 mL of MeOH, 5.2 g of 6-amino-hexanoic acid, and 875 mL of ddH2O. 6. Tween-20. 7. PBST: Add 0.5 mL of Tween-20 into 1000 of mL PBS. 8. PBST-milk: Dissolve 5 g of nonfat milk powder into 100 mL of PBST. 9. Antibodies: Phospho-PKA substrate (RRXS*/T*) rabbit mAb (Cell Signaling Technology, 9624S); mouse anti HA-tag mAb (ABclonal, AE008); mouse anti GFP-tag mAb (ABclonal, AE012). 2.4

Equipment

1. Ultrapure water purification system. 2. Biological safety cabinet. 3. CO2 incubator. 4. Fluorescence microscope. 5. Water bath. 6. Ice machine. 7. SDS-PAGE apparatus. 8. Shaker. 9. Bio-Rad Trans-Blot Turbo transfer system. 10. Gel imager (e.g., Bio-Rad ChemiDoc). 11. Opera Phenix Plus High Content Screening System.

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Methods

3.1 Design of Bioorthogonal Ligand Tethering

1. Choose a structure to guide the design. PKA-Cα is used as the model enzyme here. A list of reported structures of human PKA-Cα can be found on UniProt (https://www.uniprot. org/uniprotkb/P17612/), including many protein-inhibitor complexes such as 2GU8, 3EQC, 3L9L, 3MVJ, 3OVV, 3P0M, 3VQH, 4UJ1, 5BX7, 5UZK, etc. The small molecule in 3MVJ can be readily functionalized with a linker and tetrazine through its extruding primary amine. Thus, we choose 3MVJ to guide the design. 2. Design of the small-molecule conjugate. The study [12] reported 3MVJ shows the IC50 of a panel of related molecules against PKA. The molecule containing a piperidine (instead of pyrrolidine in 3MVJ) is more potent in inhibiting PKA activity. Hence, we derive the small-molecule conjugate Tet-PKA (Fig. 3) from this compound.

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3. Identify sites for BCN incorporation. In the structure of 3MVJ, several solvent-exposed amino acid residues surround the extruding primary amine. These residues are potential tethering sites, and we randomly choose G53, Q85, and K193 for BCN incorporation (Fig. 2). 3.2 Synthesis of the Small-Molecule Conjugate Tet-PKA 3.2.1

Intermediate A1

Tet-PKA contains a tetrazine group for bioorthogonal reaction with bicyclo[6.1.0]nonyne and an adenine derivative for kinase inhibition. The conjugate is synthesized from commercially available chemicals in six steps as shown in Fig. 3. 1. Add 94 mg of 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine (0.50 mmol, 1.0 eq), 100 mg of (R)-3-(Boc-amino)piperidine (0.50 mmol, 1.0 eq), 210 mg of sodium bicarbonate (2.5 mmol, 5.0 eq), and 5 mL of ethanol into a 25 mL round-bottom flask with a magnetic stir bar. 2. Place a reflux condenser (with flowing cooling water or similar) on top of the round-bottom flask. 3. Use an oil bath (or similar) to heat the reaction mixture. Set the temperature of the oil bath at 80 °C. At this temperature, ethanol in the round-bottom flask should boil, and the resulting vapor condenses in the reflux condenser, dropping back to the round-bottom flask. Keep the reaction mixture under reflux for 10 h. At this stage, the complete consumption of 5-dichloro-7H-pyrrolo[2,3-d]pyrimidine, as well as the formation of A1, can be monitored by TLC (EtOAc/petroleum ether 1:1). 4. Cool the reaction mixture to room temperature, and then remove the solvent (see Note 6). 5. Dissolve the residue in a mixture of water (10 mL) and EtOAc (50 mL), and pour the mixture into a separating funnel. 6. Get rid of the bottom aqueous layer. Add 25 mL of brine, and vigorously shake the separating funnel. Wait for the separation of the two layers. Discard the bottom aqueous layer. Collect the upper organic layer and add some anhydrous Na2SO4 to absorb the remaining water in EtOAc. Filter the solution and concentrate the filtrate (see Note 6). 7. Purify the material by silica gel column chromatography using 2% MeOH in CH2Cl2 to afford tert-butyl (R)-(1-(5-chloro7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-3-yl)carbamate A1 as a white solid (135 mg, 77%). 1H NMR (400 MHz, CDCl3) δ 10.97 (s, 1H), 8.36 (s, 1H), 7.14 (s, 1H), 5.22 (d, J = 7.6 Hz, 1H), 3.89–3.83 (m, 2H), 3.75–3.71 (m, 1H), 3.62–3.50 (s, 2H), 1.94–1.88 (m, 2H), 1.76–1.72 (m, 2H), 1.43 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 159.8, 155.2, 151.2, 150.5, 120.0, 103.8, 103.6, 79.2, 53.2, 51.6, 46.7, 30.2, 28.4, 22.7. ESI-(+)-HRMS (M+H)+ calculated for C16H23ClN5O2: 352.1535; found: 352.1525.

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Intermediate A2

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1. Place 106 mg of A1 (0.30 mmol, 1.0 eq) and a magnetic stir bar in a 25 mL round-bottom flask. 2. Slowly add 6 mL of 4 N HCl in dioxane (24 mmol, 80 eq) into the round-bottom flask and keep the reaction mixture at the room temperature for 1 h. 3. Remove the solvent under reduced pressure (see Note 6) to afford (R)-1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl) piperidin-3-amine hydrochloride A2 as a white solid (82 mg, 95% yield).

3.2.3

Intermediate A3

1. Add 58 mg of A2 (0.20 mmol, 1.0 eq), 52 μL of DIPEA (0.30 mmol, 1.5 eq), 53 μL of EDC (0.30 mmol, 1.5 eq), and 2 mL of dry DMF into a 25 mL round-bottom flask with a magnetic stir bar for constant agitation. 2. Cool the solution to 0 °C (e.g., ice bath) and add 51 mg of NBOC-6-aminocaproic acid (0.22 mmol, 1.1 eq) with constant agitation. 3. Return the reaction mixture to room temperature and stirring for 18 h. 4. Dilute the reaction mixture in EtOAc (50 mL), add 25 mL of brine, and vigorously shake the separating funnel. Wait for the separation of the two layers. Discard the bottom aqueous layer. Collect the upper organic layer and add some anhydrous Na2SO4 to absorb the remaining water in EtOAc. Filter the solution. 5. Remove the solvent (see Note 6) and purify the material by silica gel column chromatography using 2% MeOH in CH2Cl2 to afford tert-butyl (R)-(6-((1-(5-chloro-7H-pyrrolo[2,3-d] pyrimidin-4-yl)piperidin-4-yl)piperidin-3-yl)amino)-6-oxohexyl)carbamate A3 as a white solid (40 mg, 43%). 1H NMR (400 MHz, CDCl3) δ 11.60 (s, 1H), 8.34 (s, 1H), 7.16 (s, 1H), 6.82 (s, 1H), 4.58 (s, 1H), 4.17 (s, 1H), 3.86 – 3.81 (m, 2H), 3.66 – 3.58 (m, 2H), 3.10 – 3.04 (m, 2H), 2.15 (t, J = 7.4 Hz, 2H), 1.91 – 1.78 (m, 3H), 1.70 – 1.66 (m, 1H), 1.63 – 1.56 (m, 2H), 1.50 – 1.44 (m, 2H), 1.42 (s, 9H), 1.34 – 1.23 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 172.4, 159.8, 156.0, 151.3, 150.7, 120.1, 103.9, 103.7, 79.1, 52.3, 52.1, 45.8, 40.4, 36.8, 29.7, 29.1, 28.4, 26.3, 25.3, 22.2. ESI-(+)HRMS (M+H)+ calculated for C22H34ClN6O3: 465.2375; found: 465.2375.

3.2.4

Intermediate A4

1. Add 46 mg of A3 (0.10 mmol, 1.0 eq) and a magnetic stir bar in a 25 mL round-bottom flask. 2. Slowly add 2 mL of 4 N HCl in dioxane (8 mmol, 80 eq) into the round-bottom flask and keep the reaction mixture at the room temperature for 1 h.

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3. Remove the solvent (see Note 6) to afford (R)-6-amino-N(1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-3yl)hexanamide hydrochloride as a white solid (39 mg, 97% yield). 3.2.5

Intermediate A5

1. Add 147 mg of 4-cyanobenzoic acid (1.00 mmol, 1.0 eq), 64 μL of CH2Cl2 (1.00 mmol, 1.0 eq), 64 mg of sulfur (2.00 mmol, 2.0 eq), and 1 mL of ethanol into a 30 mL microwave reaction tube with a magnetic stir bar. 2. Slowly add 0.4 mL of hydrazine monohydrate (8.00 mmol, 8.0 eq) into the tube and keep the reaction mixture at 50 °C in the microwave reactor for 24 h. 3. Add 3 mL of CH2Cl2 and 690 mg of sodium nitrite (10 mmol, 10 eq) in 10 mL of H2O into the reaction mixture. 4. Slowly add 3.4 mL of acetic acid (60 mmol, 60 eq) during which the solution turns bright red. Pour the mixture into a separating funnel. 5. Add 30 mL of EtOAc into the mixture, and vigorously shake the separating funnel. Wait for the separation of the two layers. Collect the upper organic layer. Repeat this step for three times. 6. Add some anhydrous Na2SO4 to the combined EtOAc fraction to absorb the remaining water. Filter the solution and remove the solvent (see Note 6). 7. Purify the material by silica gel column chromatography using 2% MeOH in CH2Cl2 to afford 4-(1,2,4,5-tetrazin-3-yl)benzoic acid as a red solid (111 mg, 55%). 1H NMR (400 MHz, DMSO-d6): δ 10.66 (s, 1H), 8.62 (d, J = 8.8 Hz, 2H), 8.22 (d, J = 8.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 166.7, 165.1, 158.3, 135.7, 134.4, 130.2, 128.0. The spectroscopic data are in accordance with the reported literature [12].

3.2.6 Small-Molecule Conjugate Tet-PKA

1. Add 7.1 mg of A5 (35 μmol, 1.0 eq), 12 μL of DIPEA (70 μmol, 2.0 eq), 20 mg of HATU (52 μmol, 1.5 eq), and 2 mL of anhydrous THF into a 25 mL round-bottom flask with a magnetic stir bar for constant agitation and stirring for 2 h. 2. Add 14 mg of A4 (35 μmol, 1.0 eq) to the mixture and stirring for 4 h. 3. Remove the solvent (see Note 6) and purify the material by silica gel column chromatography using 5% MeOH in CH2Cl2 to afford Tet-PKA compound as a red solid (12 mg, 62%). 1H NMR (400 MHz, DMSO-d6) δ 12.12 (s, 1H), 10.64 (s, 1H), 8.70 (t, J = 5.6 Hz, 1H), 8.57 (d, J = 8.4 Hz, 2H), 8.23 (s, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.81 (J = 7.6 Hz, 1H), 7.44 (s, 1H), 4.10 – 4.01 (m, 2H), 3.85 – 3.77(m, 1H), 3.32 – 3.27

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(m, 2H), 3.03 – 2.96 (m, 1H), 2.85 (dd, J = 12.6, 9.8 Hz, 1H), 2.08 (t, J = 7.4 Hz, 2H), 1.91 – 1.87 (m, 1H), 1.81 – 1.77 (m, 1H), 1.71 – 1.64 (m, 1H), 1.60 – 1.49 (m, 4H), 1.44 – 1.40 (m, 1H), 1.34 – 1.29 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 171.6, 165.4, 165.2, 158.8, 158.2, 151.0, 150.8, 138.3, 134.1, 128.2, 127.7, 121.0, 102.6, 101.5, 53.5, 49.9, 45.5, 35.4, 30.2, 28.8, 26.1, 25.1, 23.6. ESI-(+)-HRMS (M+H)+ calculated for C26H30ClN10O2: 549.2236; found: 549.2245. 3.3

Cell Culture

1. To revive HEK293T cells, thaw one tube of frozen cell stock in a water bath at 37 °C. Dilute 1 mL cell stock with 4 mL of pre-warmed growth media in a 15 mL tube. Spin down cells at 300 × g for 5 min. Discard the supernatant to remove DMSO from the media. Resuspend cells in 5 mL pre-warmed culture media and transfer to a T25 cell culture flask. Maintain cells at 37 °C and 5% CO2. 2. Passage cells when they reach ~80% confluence. Remove culture media; rinse cells with 1 mL of pre-warmed PBS to remove residual serum which can inactivate trypsin. Add 0.5 mL of pre-warmed 0.25% trypsin and incubate cells at 37 °C for 1 min or until the cells have detached. 3. Add 2.5 mL of growth media to deactivate trypsin; separate cells by gently pipetting up and down ten times. Dilute 0.5 mL of resuspended cells to 4.5 mL of warm complete media in a new T25 flask (1:6 dilution). Incubate cells at 37 ° C and 5% CO2. 4. Passage cells every 2 days when cells reach ~80% confluency. Examine the cell morphology under a microscope. Cells are discarded after 25 passages.

3.4

Transfection

1. Take about 0.5 mL of 0.01% poly-lysine solution to rinse each well in the 24-well plate, and then wash each well with PBS (see Note 7). 2. When cells reach ~80% confluency in a T25 flask, detach cells with 0.5 mL of 0.25% trypsin and 4.5 mL of growth media. Mix 100 μL of resuspended cells with 400 μL of pre-warmed growth media. Add the mixture (around 1.5 × 105 cells) to a single well of the 24-well culture plate. Swirl the plate in one direction. Incubate the cells at 37 °C and 5% CO2 overnight. 3. When the cells reach ~80% confluency, replace growth media with pre-warmed 0.1% FBS in DMEM (vol/vol; see Note 8) supplemented with or without 0.1 mM BCNK (see Note 9). Place the plate back in the incubator. 4. Prepare the transfection mixture following the manufacture’s suggestion. For a single well of 24-well culture plate, prepare

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two tubes containing 25 μL of Opti-MEM each, add plasmids (300 ng of pPylRS(AF), 300 ng of pPKA-Cα, and 200 ng of pGFP-LRRATLVD or pExRai-AKAR2; see Note 10) to one tube and 1.5 μL of Lipofectamine 2000 reagent to the other tube, and then mix them to obtain the mixture. After ~15-min incubation at room temperature, the resulting mixture is added to the corresponding well of plate. Gently mix by swirling the plate in one direction. 5. Incubate the cells at 37 °C and 5% CO2 for 24 h, and then check the expression under fluorescence microscope. Normally, over 70% of cells show green fluorescence. 3.5 Detection of Kinase Inhibition by Immunoblotting

1. Remove the media, and then rinse cells with pre-warmed PBS twice to remove residual BCNK, which can react with TetPKA. Do the wash steps carefully to minimize detachment of cells from the surface. 2. Add 0.5 mL of pre-warmed DMEM with 0.01% FBS to each well. Add 5 μL of 1 mM Tet-PKA stock solution to each well to reach the final concentration of 10 μM (see Note 11). Incubate the cells at 37 °C and 5% CO2 for 2 h. 3. Prepare the protein extraction buffer. We use RIPA buffer containing protease and phosphatase inhibitor cocktails to lyse cells. 4. Remove media, and then rinse the cells with PBS once. Add 50 μL of protein extraction buffer to each well, and keep the plate on ice for 10 min. Bend a 200 μL pipette tip and use it to scrape cells from the surface. Transfer all content from the well to a 1.5 mL tube. Centrifuge at 20,000 × g and 4 °C for 10 min. 5. Prepare new tubes containing 16 μL of SDS-PAGE sample loading buffer. Take 48 μL of supernatant from step 4 to the new tube; gently mix by pipetting up and down. Heat the samples at 95 °C for 5 min to denature protein. If the samples are to be used at a later date, they should be stored at -80 °C. 6. For the detection of PKA-Cα (~40 kDa) and the reporter (~27 kDa), a gel with 12.5% of acrylamide is recommended. Assemble the electrophoresis apparatus and set the gels. Load the sample and markers to the well of SDS-PAGE gel. Run the samples at a constant volt (200 V is recommended) until bromophenol blue (the loading dye in LDS buffer) comes to the bottom of the gel. Disassemble the electrophoresis apparatus and take out the gels. Carefully transfer the gel to a container with running buffer. 7. There are three types for transferring proteins on a gel to a membrane: wet, semi-dry, and dry types. We choose the semidry type by using the Bio-Rad Trans-Blot Turbo transfer

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system. Firstly, soak two sheets of filter papers in anode buffer 1, and then put them in the bottom part of transfer cassette. Secondly, soak one sheet of filter paper in anode buffer 2 and then put then on the first two filter papers. Put nitrocellulose membrane on the top, and then spread the gel on membrane gently. Finally, soak two sheets of filter papers in cathode buffer and put them on the gel. 8. Load the transfer cassette into the transfer device. For Bio-Rad TransBlot, successively click list-BioRad-one mini gelStandard-A run for 1 to 2 gels. The current and time are 0.5 A and 30 min, respectively. 9. Rinse the membranes in PBST-milk solution when transfer is done, and then shake the container for 1 h for blocking. 10. Prepare the primary antibody solution. Two primary antibodies, anti HA antibody and anti Phospho-PKA substrate (RRXS*/T*), are diluted to 1:1000 with PBST-milk. Transfer the membranes to the primary antibody solutions and shake the containers continuously at 4 °C overnight. 11. The next day, wash the membrane three times with PBST for 10 min. Dilute the corresponding secondary to 1:20,000 with PBST-milk. Transfer the membrane to the secondary antibody solution and shake the container continuously at room temperature for 1 h. 12. Wash the membrane three times with PBST for 10 min, and then immerse it in ECL solution. Place the membrane on a thin flat plate and image the membrane by using imager (Fig. 4). 3.6 High-Content Live Cell Imaging

To monitor the change in catalytic activity of PKA-Cα at actual time, an excitation-ratiometric PKA sensor, ExRai-AKAR2 [14], is employed here. This fluorescent sensor is a variant of GFP and can be excited with 405 or 480 nm. Excitation with 405 nm leads to fluorescence at 530 nm in the absence of active PKA-Cα, whereas active PKA-Cα leads to high 530-nm fluorescence by 480-nm excitation. Thus, the ratio of 530-nm fluorescence upon 480-nm excitation over upon 405-nm excitation correlates with PKA-Cα activity (i.e., high 480/405 ratio in the presence of active PKA-Cα). 1. Treat each well of 96-well culture plate (e.g., Cellcarrier 96-ultra) with 0.01% poly-lysine solution, and then wash each well with fresh PBS solution once. 2. Maintain cells in a T25 flask. Detach cells with 0.5 mL of 0.25% trypsin and 4.5 mL of complete media when cells reach ~80% confluency. 3. Mix 30 μL of resuspended cells from step 1 with 400 μL of pre-warmed complete media, and then seed the cells (around

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Fig. 4 iBOLT of PKA-Cα using Tet-PKA. (a) HEK293T cells expressing a constitutively active PKA-Cα bearing a BCNK at position 53, 85, or 193 were treated with or without Tet-PKA (10 μM). Phosphorylation of its substrate and full-length PKA-Cα were detected via immunoblotting. (b) HEK293T cells expressing PKA-Cα with or without a BCNK at 193 position were treated with Tet-PKA at the indicated concentrations. (c) HEK293T cells expressing PKA-Cα(193BCNK) were treated with Tet-PKA (10 μM), and phosphorylation of probe was represented by the ratio of 512-nm fluorescence exited by 488 nm and 405 nm via high-content screening system. The curve shows the change of normalized 488/405 ratio against time. Images show cells at different time points after addition of Tet-PKA (i.e., post inhibition), where the brightness of cells represents the 488/405 ratio

5 × 104 cells) in a single well of the prepared 96-well culture plate. Then incubate the cells at 37 °C and 5% CO2 overnight. 4. Co-transfect cells with plasmids encoding PylRS, PKA-Cα, and probe as described in Subheading 3.2 transfection section. Incubate the cells at 37 °C and 5% CO2 for at least 24 h. 5. Check the expression under fluorescence microscope. Normally, over 70% of cells show green fluorescence. Then, wash the cells with PBS or serum-free DMEM twice to remove excess BCNK. Moreover, prepare the Tet-PKA in proper concentration using serum-free DMEM (see Note 12). 6. Turn on the Opera Phenix Plus High Content Screening System and the connected computer. Open the corresponding software, Harmony v5.1, log in and set the environmental factor to 37 °C and 5% CO2 (Settings-Environmental control). Open the carbon dioxide gas valve. 7. Click “Eject,” then put the culture plate on the carrier, and then click “Load.” 8. Set up a new experiment. Our plate type is “Cellcarrier 96-ultra,” and we choose “Two Peak (default)” in autofocus options, “20× water, NA = 1.0” in objective options and “confocal” mode. In channel selection options, our channel group one is “488 - Intensity Nucleus Cerulean,” channel

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group two is “extended – 405.” Then click “Layout selection” – “stack” to set up the Z-axis parameter. Generally, we use a combination of “First plane at 10 μm,” “number of planes is 20,” and “distance is 1 μm.” In “Navigation” – “Defined Layout” option, select a single well, and then select target wells in the bellowed “Well” option. 9. Start “test” and see the resulting imagines in “Navigation” – “test images.” This step is essential for the following measurement. As there are two channels in our experiment, it is necessary to adjust the “height” of each channel to allow their resulting imagine to be fully overlapped (see Note 13). 10. Set up time series. Here we set two sequences and one break. The first sequence allows us to measure the initial activity. Followed break allows us to add ligand Tet-PKA to culture media. Then we monitor the change of PKA activity in sequence two. 11. Start the measurement. 12. After the measurement, set up evaluation file to calculate the ratio of two channels. Here we use the ratio of “488 - Intensity Nucleus Cerulean” to “extended – 405” to reflect the activity of PKA (Fig. 4).

4

Notes 1. Tet-PKA is normally stable in the DMSO stock solution at 20 °C for several months. If in doubt, check the integrity of the stock solution by LC-MS. 2. We use the commercial human ORF clone of PRKACA from Horizon Discovery as the PCR template to construct PKA-Cα variants containing a Kozak sequence at the 5′-end and a C-terminal HA tag. The DNA sequence of PKA-Cα (193TAG) is shown below: GCCACCATGGGCAACGCCGCCGCCGCCAAAAGG GCAGCGAGCAGGAGAGCGTGAAAGAATTCTTAGCCA AAGCCAAAGAAGATTTTCTTAAAAAATGGGAAAGTCC CGCTCAGAACACAGCCCACTTGGATCAGTTTGAACG AATCAAGACCCTCGGCACGGGCTCCTTCGGGCGGGT GATGCTGGTGAAACACAAGGAGACCGGGAACCACTA TGCCATGAAGATCCTCGACAAACAGAAGGTGGTGAAA CTGAAACAGATCGAACAGACCCTGAATGAAAAGCGCA TCCTGCAAGCTGTCAACTTTCCGTTCCTCGTCAAACT CGAGTTCTCCTTCAAGGACAACTCAAACTTATACATG GTCATGGAGTACGTGCCCGGCGGGGAGATGTTCTCA CACCTACGGCGGATCGGAAGGTTCAGTGAGCCCCAT GCCCGTTTCTACGCGGCCCAGATCGTCCTGACCTTT

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GAGTATCTGCACTCGCTGGATCTCATCTACAGGGACC TGAAGCCGGAGAATCTGCTCATTGACCAGCAGGGCT ACATTCAGGTGACAGACTTCGGTTTCGCCAAGCGTG TATAGGGCAGGACTAGGACCTTGTGCGGCACCCCTG AGTACCTGGCCCCTGAGATTATCCTGAGCAAAGGCTA CAACAAGGCCGTGGACTGGTGGGCCCTGGGGGTTCT TATCTATGAAATGGCCGCTGGCTACCCGCCCTTCTTC GCAGACCAGCCCATCCAGATCTATGAGAAGATCGTCT CTGGGAAGGTGCGCTTCCCTTCCCACTTCAGCTCTG ACTTGAAGGACCTGCTGCGGAACCTCCTGCAGGTAG ATCTCACCAAGCGCTTTGGGAACCTCAAGAATGGGG TCAACGATATCAAGAACCACAAGTGGTTTGCCACAAC TGACTGGATTGCCATCTACCAGAGGAAGGTGGAAGC TCCCTTCATACCAAAGTTTAAAGGCCCTGGGGATACG AGTAACTTTGACGACTATGAGGAAGAAGAAATCCGGG TCTCCATCAATGAGAAGTGTGGCAAGGAGTTTTCTGA GTTTGGATCCTACCCATACGATGTTCCAGATTACGCT TGA. 3. We use the commercially available BCNK (CAS: 1384100-451) from SiChem (SC-8016). BCNK is not that soluble in water, but its sodium salt has good aqueous solubility. Thus, we use 0.1 M NaOH solution to prepare 0.1 M BCNK stock solution. The stock solution is stored at 4 °C. 4. Commercially available RIPA may have different composition, affecting lysis efficiency. We use the one from Merck (R0278). 5. There are many commercial choices for the sample loading buffer. We add 4% (vol/vol) β-mercaptoethanol into 4 × LDS loading buffer from Thermo Fisher (NP0008) for preparing SDS-PAGE samples. 6. We remove solvents at 30–40 °C under reduced pressure at using a rotary evaporator. If the solution contains HCl, the rotary evaporator needs to be in a fume hood or its exhaust is quenched by NaHCO3(aq). 7. Poly-lysine can enhance the attachment between the cells and culture plate. Thus, pre-treating the plate with poly-lysine can avoid cells from detaching from the plate in wash steps. However, a high level of poly-lysine is toxic to cells, so removal of residual poly-lysine by washing with PBS is required. For cells (e.g., HeLa) that firmly attach to plastic surface, it is not necessary to pre-treat the plates. 8. Serum contains growth factors that activate different kinase signaling pathways. Thus, to minimize background signals from endogenous kinases (i.e., PKA in this case), low serum media are used. 9. If an experiment requires six wells to be cultured in the presence of BCNK, add 3 μL of 100 mM BCNK into 3 mL of 0.1%

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FBS in DMEM, mix, and dispense 0.5 mL of the solution into each well for a total of six wells. 10. The ratio of three plasmids for co-transfection is not fixed. If BCN incorporation efficiency is low for a particular site, increase the amount of pPylRS(AF) and/or pPKA-Cα may improve the outcome. 11. We perform incubation with 10 μM of tetrazine conjugate (i.e., Tet-PKA in this case) for 2 h as the standard condition. However, inhibition can be achieved with a lower concentration of tetrazine conjugate and less time. 12. We recommend to prepare Tet-PKA in a concentration of 30 μM. As the volume of culture media for each well on 96-well culture plate is 100 μL here, add 50 μL of prepare Tet-PKA solution to reach our target concentration (10 μM). 13. Another aim of this test is to adjust the “time” and “power” of each channel according to fluorescence intensity showed in “Image Control.” The maximum of intensity should between 1,200 and 68,000.

Acknowledgements This work was supported by the Shenzhen Bay Laboratory and National Natural Science Foundation of China. We thank Dingding Mo for preliminary works of this project. We are grateful to Simon Els€asser and Jin Zhang for sharing the sequence of pPylRS (AF) and pExRai-AKAR2, respectively. We also thank SZBL Mass Spectrometry Facility and SZBL Bioimaging Core for assistance. References 1. Sreedhar A, Zhao Y (2018) Dysregulated metabolic enzymes and metabolic reprogramming in cancer cells. Biomed Rep 8(1):3–10. https://doi.org/10.3892/br.2017.1022 2. Dantuma NP, Bott LC (2014) The ubiquitinproteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution. Front Mol Neurosci 7:70. https://doi. org/10.3389/fnmol.2014.00070 3. Dhanwani R, Takahashi M, Sharma S (2018) Cytosolic sensing of immuno-stimulatory DNA, the enemy within. Curr Opin Immunol 50:82–87. https://doi.org/10.1016/j.coi. 2017.11.004 4. Islam K (2018) The bump-and-hole tactic: expanding the scope of chemical genetics. Cell Chem Biol 25(10):1171–1184. https:// doi.org/10.1016/j.chembiol.2018.07.001

5. Shogren-Knaak MA, Alaimo PJ, Shokat KM (2001) Recent advances in chemical approaches to the study of biological systems. Annu Rev Cell Dev Biol 17:405–433. https:// doi.org/10.1146/annurev.cellbio.17.1.405 6. El-Brolosy MA, Stainier DYR (2017) Genetic compensation: a phenomenon in search of mechanisms. PLoS Genet 13(7):e1006780. https://doi.org/10.1371/journal.pgen. 1006780 7. Schreiber SL, Kotz JD, Li M, Aube J, Austin CP, Reed JC, Rosen H, White EL, Sklar LA, Lindsley CW, Alexander BR, Bittker JA, Clemons PA, de Souza A, Foley MA, Palmer M, Shamji AF, Wawer MJ, McManus O, Wu M, Zou B, Yu H, Golden JE, Schoenen FJ, Simeonov A, Jadhav A, Jackson MR, Pinkerton AB, Chung TD, Griffin PR, Cravatt BF, Hodder PS, Roush

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WR, Roberts E, Chung DH, Jonsson CB, Noah JW, Severson WE, Ananthan S, Edwards B, Oprea TI, Conn PJ, Hopkins CR, Wood MR, Stauffer SR, Emmitte KA, Team NMLP (2015) Advancing biological understanding and therapeutics discovery with small-molecule probes. Cell 161(6):1252–1265. https://doi.org/10. 1016/j.cell.2015.05.023 8. Tsai YH, Doura T, Kiyonaka S (2021) Tethering-based chemogenetic approaches for the modulation of protein function in live cells. Chem Soc Rev 50(14):7909–7923. https:// doi.org/10.1039/d1cs00059d 9. Tsai YH, Essig S, James JR, Lang K, Chin JW (2015) Selective, rapid and optically switchable regulation of protein function in live mammalian cells. Nat Chem 7(7):554–561. https:// doi.org/10.1038/nchem.2253 10. Spear LA, Huang Y, Chen J, No¨dling AR, Virdee S, Tsai YH (2022) Selective inhibition of cysteine-dependent enzymes by bioorthogonal tethering. J Mol Biol 434(8):167524. h ttps://doi.o rg/1 0.1016/j.jmb.2022 . 167524 11. No¨dling AR, Spear LA, Williams TL, Luk LYP, Tsai YH (2019) Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells. Essays Biochem 63(2):237–266. https://doi.org/10.1042/ ebc20180042 12. Freeman-Cook KD, Autry C, Borzillo G, Gordon D, Barbacci-Tobin E, Bernardo V, Briere D, Clark T, Corbett M, Jakubczak J, Kakar S, Knauth E, Lippa B, Luzzio MJ, Mansour M, Martinelli G, Marx M, Nelson K, Pandit J, Rajamohan F, Robinson S, Subramanyam C, Wei L, Wythes M, Morris J (2010) Design of selective, ATP-competitive inhibitors of Akt. J Med Chem 53(12): 4615–4622. https://doi.org/10.1021/ jm1003842

13. Zhang J, Hupfeld CJ, Taylor SS, Olefsky JM, Tsien RY (2005) Insulin disrupts betaadrenergic signalling to protein kinase a in adipocytes. Nature 437(7058):569–573. https:// doi.org/10.1038/nature04140 14. Zhang JF, Liu B, Hong I, Mo A, Roth RH, Tenner B, Lin W, Zhang JZ, Molina RS, Drobizhev M, Hughes TE, Tian L, Huganir RL, Mehta S, Zhang J (2021) An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice. Nat Chem Biol 17(1): 39–46. https://doi.org/10.1038/s41589020-00660-y 15. Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S (2008) Multistep engineering of pyrrolysyltRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol 15(11):1187–1197. https://doi.org/10. 1016/j.chembiol.2008.10.004 16. Bartoschek MD, Ugur E, Nguyen TA, Rodschinka G, Wierer M, Lang K, Bultmann S (2021) Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells. Nucleic Acids Res 49(11):e62. https://doi. org/10.1093/nar/gkab132 17. Schmied WH, Elsasser SJ, Uttamapinant C, Chin JW (2014) Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/ tRNA expression and engineered eRF1. J Am Chem Soc 136(44):15577–15583. https:// doi.org/10.1021/ja5069728 18. Mills EM, Barlow VL, Jones AT, Tsai YH (2021) Development of mammalian cell logic gates controlled by unnatural amino acids. Cell Rep Methods 1(6):100073. https://doi.org/ 10.1016/j.crmeth.2021.100073

Chapter 17 Site-Specific Incorporation of Sulfotyrosine into Proteins in Mammalian Cells Xinyuan He, Yan Chen, Jiantao Guo, and Wei Niu Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in numerous extracellular protein-protein interactions. It is involved in diverse physiological processes and the development of human diseases, including AIDS and cancer. To facilitate the study of PTS in live mammalian cells, an approach for the site-specific synthesis of tyrosine-sulfated proteins (sulfoproteins) was developed. This approach takes advantage of an evolved Escherichia coli tyrosyl-tRNA synthetase to genetically encode sulfotyrosine (sTyr) into any proteins of interest (POI) in response to a UAG stop codon. Here, we give a step-by-step account of the incorporation of sTyr in HEK293T cells using the enhanced green fluorescent protein as an example. This method can be widely applied to incorporating sTyr into any POI to investigate the biological functions of PTS in mammalian cells. Key words Protein tyrosine O-sulfation (PTS), Sulfotyrosine incorporation, Genetic code expansion, Amber suppression, Green fluorescent protein, Mammalian cells

1

Introduction Protein tyrosine O-sulfation (PTS) is a posttranslational modification (PTM) that occurs especially in eukaryotic cells [1–3]. It is catalyzed by the membrane-bound tyrosylprotein sulfotransferases (TPSTs) in the trans-Golgi through the transfer of an activated sulfate group from 30 -phosphoadenosine 50 -phosphosulfate to a protein tyrosine residue (Fig. 1). The integration of inorganic sulfate groups into protein molecules plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes, including viral infection, chemotaxis, and immune responses [4, 5]. Despite its importance, PTS has been challenging to study due to the lack of methods to prepare target proteins in a homogeneously sulfated state [6, 7]. To circumvent this challenge, synthetic approaches have been developed to chemically synthesize sulfopeptides to study protein-ligand interactions [8–

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_17, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 A comparison of protein tyrosine O-sulfation (PTS) and the translational synthesis of sulfoprotein via genetic code expansion. (a) Structure of sTyr. (b) Molecular mechanism of protein tyrosine O-sulfation. The tyrosine residue of a substrate protein is sulfated by a tyrosylprotein sulfotransferase (TPST) using the sulphate donor, adenosine 30 -phosphate 50 -phosphosulfate (PAPS). PAP, adenosine 30 , 50 -diphosphate. (c) Scheme of sTyr incorporation into a protein in mammalian cells using an amber stop codon and engineered tyrosyl-tRNA synthetase (sTyrRS). The sTyrRS aminoacylates BstRNATyrCUA with sTyr and the charged tRNA decodes the UAG amber codon in a protein of interest

10]. However, synthetic approaches are often limited to small peptides. In 2006, Liu et al. engineered a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS) to genetically incorporate sulfotyrosine (sTyr) into proteins in response to amber UAG codon [11]. The genetic code expansion of sTyr allows the synthesis of full-length sulfoproteins in Escherichia coli. Nevertheless, this method is limited to bacterial hosts and cannot be applied in mammalian cells due to cross-reactivity of the genetic encoding system. To synthesize site-specifically sulfated proteins in live mammalian cells, we have engineered tyrosyl-tRNA synthetases that can co-translationally incorporate sTyr in response to amber UAG codons in mammalian cells [12]. This approach allows the site-specific insertion of sTyr in any proteins of interest (Fig. 1c). In this chapter, we describe a step-by-step procedure for the incorporation of sTyr at position 40 of the target protein, enhanced green fluorescent protein (EGFP), in HEK293T cells using a dualplasmid system (Fig. 2). On one plasmid, a sTyr-specific tRNA synthetase (sTyrRS) is expressed under the control of a CMV promoter, and a UAG-decoding tRNA (BstRNATyrCUA) is expressed from the U6 promoter (Fig. 2a). On the second plasmid, EGFP with a TAG mutation at position 40 is expressed under the CMV promoter (Fig. 2b). The two plasmids are transiently co-transfected into HEK293T cells. In the presence of sTyr, sTyrRS specifically aminoacylates BstRNATyrCUA with sTyr, and the

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B CMV

sTyrRS

psTyrRS U6

CMV

EGFP 40TAG

pEGFP

BstRNATyrCUA

Fig. 2 Schematic illustration of the expression plasmids. (a) Expression plasmid for sTyrRS and BstRNATyrCUA. sTyrRS is overexpressed under the CMV promoter, and BstRNATyrCUA is expressed by the U6 promoter. (b) Expression plasmid for EGFP-40TAG. EGFP with an amber mutation at position 40 is overexpressed by the CMV promoter. A 6His tag is expressed at the C terminus. EGFP-40TAG can be replaced by any proteins of interest for the site-specific incorporation of sTyr

Fig. 3 Fluorescence analysis of sTyr incorporation into EGFP in HEK293T cells. (a) Confocal images. HEK293T cells were co-transfected with psTyrRS and pEGFP in the absence and presence of sTyr (1 mM). The left panel shows fluorescent images, the middle panel shows brightfield images, and the right panel shows composite images of fluorescent and brightfield images. Scale bars, 10 μm. (b) Flow cytometry analysis. The normalized fluorescence was calculated by multiplying the mean fluorescence intensity by the percentage of fluorescent cells. Data are plotted as the mean  standard deviation (s.d.) from three independent experiments

charged tRNA is able to decode the UAG amber codon at position 40 in EGFP. This amber suppression results in the expression of the full-length EGFP, which is detected by confocal microscope (Fig. 3a) and is statistically quantified using flow cytometry (Fig. 3b).

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Materials All commercial chemicals are of reagent grade or higher. All solutions are prepared using Ultrapure water.

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Sulfotyrosine

Sulfotyrosine is synthesized by following our previously published procedure [13]. 1. Add 50 mL of trifluoroacetic acid and 10 g of L-tyrosine (55.6 mmol) into a dry round-bottom flask with a magnetic stir bar at 10  C. 2. While stirring under nitrogen protection, add 5 mL of chlorosulfonic acid (75.2 mmol) into the flask for over 2 min. Let the reaction run for another 5 min. 3. Add 3 mL of ethanol to quench the reaction, and stir for 2 min at room temperature. 4. Precipitate sulfotyrosine with 175 mL of diethyl ether, filter, and wash three times with 75 mL of diethyl ether. Dry the product under vacuum to remove residual ether. 5. 100 mM sTyr stock solution: Dissolve 261.3 mg of sTyr in 10 mL of H2O; sterilize the solution by filtration with a 0.22 μm sterile membrane filter (see Note 1). Store the solution at 20  C. The solution can be diluted in DMEM+10% FBS to 1 mM just before use (see Note 2).

2.2

Cell Line

1. HEK293T cells.

2.3

Plasmids

1. psTyrRS: The plasmid is based on pcDNA3.1 vector. This plasmid allows efficient expression of sTyrRS (see Note 3) under the CMV promoter and BstRNATyrCUA (see Note 4) under the U6 promoter (see Fig. 2a; see Note 5 for DNA sequences of sTyrRS and BstRNA). 2. pEGFP: This plasmid contains an EGFP-encoding gene with an amber mutation at position 40 (EGFP-40TAG) under the CMV promoter. The EGFP-40TAG can be replaced by any proteins of interest (see Note 6) for the site-specific incorporation of sTyr (see Fig. 2b; see Note 5 for DNA sequence of EGFP-40TAG).

2.4 Media and Reagents

1. Fetal bovine serum (FBS). 2. DMEM+10% FBS: Mix 450 mL of DMEM with 50 mL of FBS, and sterilize the mixture by filtration through a 0.22 μm sterile membrane. 3. 0.05% trypsin. 4. DPBS. 5. Lipofectamine 2000 transfection reagent. 6. 16% formaldehyde solution.

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7. 4% formaldehyde solution: Mix 30 mL of DPBS with 10 mL of 16% formaldehyde. The solution can be stored at 4  C for up to 1 month. 8. Penicillin-streptomycin (10,000 U/mL). 2.5

Equipment

1. Ultrapure water purification system. 2. Biological safety cabinet. 3. CO2 incubator. 4. Cell culture flasks (T25). 5. 24-well cell culture plate. 6. 0.2 μm media filter unit. 7. 35 μm cell strainers. 8. Drummond Pipet-Aid XP (or equivalent) and serological pipets (5, 10, and 25 mL). 9. Pipettes and tips (1000, 200, and 20 μL). 10. Confocal microscope (Nikon A1R-Ti2 or equivalent). 11. Flow cytometer (Beckman Coulter Cytek DxP10 or equivalent).

3

Methods

3.1 Revive and Maintain Cell Culture

1. To revive HEK293T cells, thaw one tube of cell stock from liquid nitrogen (see Note 7). Dilute 1 mL of cell stock with 4 mL of pre-warmed DMEM+10% FBS (see Note 8) in a T25 flask. Incubate cells at 37  C and 5% CO2 for 16–24 h. 2. At 16–24 h, examine cells under a microscope. More than 90% of cells should attach to the surface of the T25 flask. Replace cell culture media with 5 mL fresh pre-warmed DMEM+10% FBS. Incubate cells at 37  C and 5% CO2 for 2–3 days until cells reach ~80% confluency. 3. To maintain good cell fitness, passage cells at around 80% confluency (see Note 9). Remove culture media; gently rinse cells once with warm DPBS. Detach cells by adding 2 mL of warm 0.05% trypsin followed by incubation at 37  C for 3 min. Separate cells by pipetting up and down gently 10 times, and then inactivate trypsin by adding 3 mL of warm DMEM+10% FBS (see Note 10). Dilute 0.5 mL of resuspended cells to 4.5 mL of warm DMEM+10% FBS in a new T25 flask (1:10 dilution). Incubate cells at 37  C and 5% CO2. 4. Passage cells every 2–3 days when cells reach ~80% confluency. Examine cells frequently under a microscope. Cells are discarded after passaging for 2 months (see Note 11).

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Transfection

1. Maintain HEK293T cells in a T25 flask (see Subheading 3.1). Detach cells using 2 mL of 0.05% trypsin and 3 mL of DMEM +10% FBS when cells reach 80% confluency. 2. Mix 100 μL of detached cells with 400 μL of warm DMEM +10% FBS (see Note 12), and inoculate the cells in a single well of the 24-well culture plate (see Note 13). 3. Incubate cells at 37  C and 5% CO2 for 16–24 h when the cell population becomes 70–80% confluent. 4. Replace culture media with warm DMEM+10% FBS supplemented with either 0 mM or 1 mM sTyr. Place the plate back in the incubator while preparing transfection mixtures. 5. For the transfection of a single well of the 24-well culture plate, mix 2 μL of Lipofectamine 2000 with 48 μL of DMEM and sit the mixture at room temperature for 5 min. Mix 500 ng of psTyrRS and 500 ng of pEGFP with 50 μL of DMEM (see Note 14). 6. Combine the diluted DNA with Lipofectamine 2000 and incubate the resulting mixture at room temperature for another 20 min (see Note 15). 7. Then, add 100 μL of DNA-Lipofectamine mixture, prepared at step 3, to a single well of the 24-well culture plate. Gently mix by swirling the plate in one direction. 8. Incubate the plate at 37  C and 5% CO2 for 24–48 h until analysis.

3.3 Confocal Imaging

1. Transfect HEK293T cells with psTyrRS and pEGFP in a 24-well cell culture plate (see Subheading 3.3). Cell culture media was supplemented with either 0 mM or 1 mM sTyr. Perform transfection in three individual wells as biological triplicates. 2. Twenty-four hours after transfection, carefully remove all culture media and wash cells with 0.5 mL of DPBS once. 3. To fix cells, add 0.5 mL of 4% formaldehyde to each well. Incubate the plate at room temperature for 20 min (see Note 16). 4. After removal of formaldehyde, wash cells with DPBS for three times. 5. The plate can be stored at 4  C for up to 24 h prior to analysis under microscope. 6. Examine the expression of green fluorescent protein under a confocal microscope. Record fluorescent and brightfield images for at least three separate regions of each well (see Note 17). 7. Export fluorescent, brightfield, and composite images together with labeled scale bars at the lower-right corner of each image (Fig. 3a).

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1. Transfect HEK293T cells with psTyrRS and pEGFP and culture cells in a 24-well cell culture plate as described in Subheading 3.3. Cell culture media was supplemented with 0 mM or 1 mM sTyr. Perform transfection in three individual wells as biological triplicates. 2. Twenty-four hours after transfection, carefully remove all culture media and wash cells with 0.5 mL of DPBS once. 3. Add 0.25 mL of 0.05% trypsin to each well, incubate at 37  C for 3 min, and detach cells by gently pipetting up and down ten times. 4. Add 0.25 mL of DMEM+10% FBS to inactivate trypsin. Resuspend cell by pipetting up and down three times (step 3 of Subheading 3.1). 5. Transfer cells to a 1.5 mL centrifuge tube. Collect cells at 200 g for 5 min using a refrigerated centrifuge at 4  C. Carefully remove supernatant (see Note 18). 6. Resuspend cells in 0.5 mL of cold DPBS at 4  C by gently pipetting up and down. Collect cells at 200 g for 5 min by centrifugation at 4  C. 7. Resuspend cells in 0.5 mL of 4% formaldehyde, and incubate at room temperature for 20 min. Remove formaldehyde after centrifugation at 200 g for 5 min. 8. Wash cells three times with 0.5 mL of cold DPBS by centrifuge at 200 g for 5 min at 4  C. 9. Resuspend cells in 0.5 mL of cold DPBS at 4  C by gently pipetting up and down. Filter cells through a cell strainer (see Note 19). 10. Cells can be stored at 4  C for up to 24 h prior to flow cytometry analysis. 11. Analyze green fluorescent protein expression of 30,000 cells using a flow cytometer. 12. Use FlowJo to analyze data. Determine GFP-positive cells using non-transfected HEK293T as the negative control. Determine the GFP-positive percentage and GFP intensity for each sample. Calculate the normalized fluorescence by multiplying GFP-positive percentage and GFP intensity (see Fig. 3b).

4

Notes 1. Since sTyr has good solubility in H2O, 100 mM sTyr stock solution can be prepared directly in water. Stock solutions of higher concentrations can be prepared using NaOH(aq). Do

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not dissolve sTyr in HCl(aq). Filtration is performed in a biosafety cabinet. Aliquots of 100 mM sTyr stock solution are stored at 20  C. 2. pH is crucial for the viability of cell culture. For sTyr stock solution in H2O, the pH is determined as ~7. And the 100 mM sTyr stock solution can be diluted directly into DMEM+10% FBS to 1 mM. However, if sTyr stock solution is made in NaOH solution, the pH of DMEM+10% FBS needs to be adjusted to pH 7 before use. 3. sTyrRS is evolved from the Escherichia coli tyrosyl-tRNA synthetase to genetically incorporate sTyr into proteins in mammalian cells. The mutation sites are L71V, W129F, and D182G [12]. 4. tRNACUA expression can be a limiting factor for the efficient unAA incorporation in mammalian cells. Therefore, a strong promoter U6 is used for tRNACUA expression. In addition, a Bacillus stearothermophilus amber suppressor tRNA (BstRNATyr CUA) is utilized for efficient mammalian expression [14]. Furthermore, multiple copies of tRNACUA can be used to improve tRNACUA expression [15–18]. 5. DNA sequences. 6. To site-specifically incorporate sTyr into any proteins of interest (POI), introduce TAG mutation into the gene of POI. Replace EGFP-40TAG with POI-TAG, resulting in the plasmid construct pPOI-TAG. Co-transfect mammalian cells with psTyrRS and pPOI-TAG to genetically synthesize POI-sTyr in mammalian cells in the presence of sTyr. 7. Immediately place one tube of cell stock into a 37  C bead bath. Bead bath is preferred because it is less likely to introduce contamination than water bath. Quickly thaw the cells by gently swirling the tube until there is no ice left in the vial. Sit the tube back into the 37  C bead bath for another 2 min before transferring cells to 4 mL of pre-warmed DMEM +10% FBS. 8. To revive and subculture HEK293T cells, antibiotics (for example, penicillin-streptomycin) can be supplemented in DMEM+10% FBS to minimize contamination. 9. To maintain good cell fitness, subculture cells when it becomes ~80% confluent. Overgrown cell culture contains unhealthy or dead cells, which leads to low transfection efficiency and will negatively affect cell-based assays. 10. To detach HEK293T cells, it is crucial to incubate cells with warm 0.05% trypsin for 3 min in the 37  C incubator. Shorter time may result in insufficient trypsinization, and longer incubation may harm cell fitness. Cell dissociation can be examined

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under a microscope. More than 90% of single cells should be observed. 11. After cell revival, do not subculture HEK293T cells for more than 2 months or 25 generations. Discard old cell culture and revive new cell culture. 12. For transfection, do not include antibiotics in DMEM +10% FBS. 13. To seed cells, dilute 600 μL detached cells with 2400 μL warm DMEM+10% FBS, and then inoculate 500 μL diluted cells in each well of the 24-well culture plate. 14. To prepare transfection mixtures, DMEM instead of DMEM +10% FBS is used. 15. To prepare transfection mixtures for six wells of cells, mix 12 μL of Lipofectamine 2000 with 288 μL of DMEM. Mix 3000 ng of psTyrRS and 3000 ng of pEGFP with 300 μL of DMEM. Then, combine the mixtures and incubate for 20 min. 16. HEK293T tends to detach from the surface easily, it is crucial to fix the cells prior to confocal imaging. After fixation, it is still recommended to wash cells carefully to minimize cell loss. 17. Record images of random fields where the cell confluency is about 70–80%. 18. Following centrifugation, carefully remove as much supernatant as possible without disturbing the cell pellet. 19. Trypsin digestion does not guarantee that all cells can be disassociated into single cells. The 35 μm cell strainers can be used to filter out cell clusters. BstRNA tggagggggacggattcgaaccgccgaacccaaagggagcggatttagagtccgccgcg tttagccacttcgctacccctcc sTyrRS ATGgcaagcagtaacttgattaaacaattgcaagagcgggggctggtagcccaggtgacg gacgaggaagcgttagcagagcgactggcgcaaggcccgatcgcgctctattgcggcttc gatcctaccgctgacagcttgcatttggggcatcttgttccattgttatgcctgaaacgctt ccagcaggcgggccacaagccggttgcggttgtaggcggcgcgacgggtctgattgg cgacccgagcttcaaagctgccgagcgtaagctgaacaccgaagaaactgttcaggag tgggtggacaaaatccgtaagcaggttgccccgttcctcgatttcgactgtggagaaaa ctctgctatcgcggcgaacaactatgactttttcggcaatatgaatgtgctgaccttcctgc gcgatattggcaaacacttctccgttaaccagatgatcaacaaagaagcggttaagcagcgtc tcaaccgtgaagatcaggggatttcgttcactgagttttcctacaacctgttgcagggttatg ggttcgcctgtctgaacaaacagtacggtgtggtgctgcaaattggtggttctgaccagtg gggtaacatcacttctggtatcgacctgacccgtcgtctgcatcagaatcaggtgtttggcc tgaccgttccgctgatcactaaagcagatggcaccaaatttggtaaaactgaaggcggcgc

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agtctggttggatccgaagaaaaccagcccgtacaaattctaccagttctggatcaacactgc ggatgccgacgtttaccgcttcctgaagttcttcacctttatgagcattgaagagatcaacgc cctggaagaagaagataaaaacagcggtaaagcaccgcgcgcccagtatgtactggcgga gcaggtgactcgtctggttcacggtgaagaaggtttacaggcggcaaaacgtattaccgaa tgcctgttcagcggttctttgagtgcgctgagtgaagcggacttcgaacagctggcgcagg acggcgtaccgatggttgagatggaaaagggcgcagacctgatgcaggcactggtcgatt ctgaactgcaaccttcccgtggtcaggcacgtaaaactatcgcctccaatgccatcaccattaa cggtgaaaaacagtccgatcctgaatacttctttaaagaagaagatcgtctgtttggtcgtttta ccttactgcgtcgcggtaaaaagaattactgtctgatttgctggaaaTAA EGFP40TAG ATGgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctgg acggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacc tagggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccca ccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaag cagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttc aaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtg aaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaag ctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcat caaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccac taccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctga gcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctgga gttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaaggggcccttcga acaaaaactcatctcagaagaggatctgaatatgcataccggtcatcatcaccatcaccat TGA

References 1. Bettelheim FR (1954) Tyrosine O-sulfate in a peptide from fibrinogen. J Am Chem Soc 76: 2838–2839. https://doi.org/10.1021/ ja01639a073 2. Kehoe JW, Bertozzi CR (2000) Tyrosine sulfation: a modulator of extracellular proteinprotein interactions. Chem Biol 7(3):R57– R61. https://doi.org/10.1016/S1074-5521 (00)00093-4 3. Moore KL (2003) The biology and enzymology of protein tyrosine O-sulfation. J Biol Chem 278(27):24243–24246. https://doi. org/10.1074/jbc.R300008200 4. Farzan M, Mirzabekov T, Kolchinsky P, Wyatt R, Cayabyab M, Gerard NP, Gerard C, Sodroski J, Choe H (1999) Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96(5):667–676. https:// doi.org/10.1016/S0092-8674(00)80577-2 5. Stone MJ, Chuang S, Hou X, Shoham M, Zhu JZ (2009) Tyrosine sulfation: an increasingly recognized post-translational modification of

secreted proteins. New Biotechnol 25(5): 299–317. https://doi.org/10.1016/j.nbt. 2009.03.011 6. Thompson RE, Liu X, Ripoll-Rozada J, Alonso-Garcia N, Parker BL, Pereira PJB, Payne RJ (2017) Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors. Nat Chem 9(9):909–917. https://doi.org/ 10.1038/nchem.2744 7. Li X, Hitomi J, Liu CC (2018) Characterization of a sulfated anti-HIV antibody using an expanded genetic code. Biochemistry 57(20): 2903–2907. https://doi.org/10.1021/acs. biochem.8b00374 8. Koeller KM, Smith MEB, Wong CH (2000) Chemoenzymatic synthesis of PSGL-1 glycopeptides: sulfation on tyrosine affects glycosyltransferase-catalyzed synthesis of the O-glycan. Bioorg Med Chem 8(5): 1017–1025. https://doi.org/10.1016/ S0968-0896(00)00041-9

Incorporation of Sulfotyrosine in Mammalian Cells 9. Kitagawa K, Aida C, Fujiwara H, Yagami T, Futaki S, Kogire M, Ida J, Inoue K (2001) Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39. J Org Chem 66(1):1–10. https:// doi.org/10.1021/jo000895y 10. Stone MJ, Payne RJ (2015) Homogeneous sulfopeptides and sulfoproteins: synthetic approaches and applications to characterize the effects of tyrosine sulfation on biochemical function. Acc Chem Res 48(8):2251–2261. https://doi.org/10.1021/acs.accounts. 5b00255 11. Liu CC, Schultz PG (2006) Recombinant expression of selectively sulfated proteins in Escherichia coli. Nat Biotechnol 24(11): 1436–1440. https://doi.org/10.1038/ nbt1254 12. He X, Chen Y, Beltran DG, Kelly M, Ma B, Lawrie J, Wang F, Dodds E, Zhang L, Guo J, Niu W (2020) Functional genetic encoding of sulfotyrosine in mammalian cells. Nat Commun 11(1):4820. https://doi.org/10.1038/ s41467-020-18629-9 13. Ju T, Niu W, Cerny R, Bollman J, Roy A, Guo J (2013) Molecular recognition of sulfotyrosine and phosphotyrosine by the Src homology 2 domain. Mol BioSyst 9(7):1829–1832. https://doi.org/10.1039/c3mb70061e

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Part III Genetic Code Expansion in Other Models

Chapter 18 Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code Wes Brown, Carolyn Rosenblum, and Alexander Deiters Abstract Conditional control of protein function in a living model organism is an important tool for studying the effects of that protein during development and disease. In this chapter, we walk through the steps to generate a small-molecule-activatable enzyme in zebrafish embryos through the incorporation of a noncanonical amino acid into the protein active site. This method can be applied to many enzyme classes, which we highlight with temporal control of a luciferase and a protease. We demonstrate that strategic placement of the noncanonical amino acid completely blocks enzyme activity, which is then promptly restored after addition of the nontoxic small molecule inducer to the embryo water. Key words Conditional control, Non-canonical amino acid, Genetic code expansion, Zebrafish, Staudinger reduction

1

Introduction Zebrafish embryo development is a complex and dynamic process that involves the precise orchestration of protein function in a fairly short window of time [1–3]. Common methods such as protein overexpression or knockdown using antisense agents do not afford the temporal control necessary to probe the dynamics of protein activity during development; thus, conditional control is an attractive feature [4–7]. Conditional control is frequently performed using transgenic Cre/LoxP lines, but this method is limited by the availability of tissue-specific promotors and does not offer complete temporal control. Chemically induced dimerization is an example of small-molecule-triggered protein activation; however, it relies on protein domain fusions and has remained largely underexploited in zebrafish [8]. In this chapter, we describe a universal method of temporally controlling an enzyme in zebrafish embryos using genetic code expansion [9, 10]. In particular, we focus on a method that installs a caging group onto an active site lysine,

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_18, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 (a) Enzyme caging through PABK insertion and temporal control through removal of the caging group (star) with a small-molecule phosphine (triangle) to restore protein activity. (b) Structures of PABK and 2DPBM and the decaging mechanism. (c) Injection of the listed components results in incorporation of PABK at the amber stop codon (UAG) in the gene of interest

blocking function, until the caging group is removed through addition of a small molecule to the embryo water, restoring the native active site (Fig. 1a) [11]. This approach relies on amber stop codon suppression using an orthogonal system derived from M. barkeri, consisting of an evolved pyrrolysyl-tRNA synthetase (PylRS) and pyrrolysyl tRNA (PylT) pair that charges the amber stop codon-recognizing tRNA with para-azidobenzyloxycarbonyl lysine (PABK, Fig. 1b) [12, 13]. The para-azidobenzyloxycarbonyl moiety is removed after phosphine-induced Staudinger reduction of the azide to an amine which triggers self-immolation through 1,6-elimination (Fig. 1c). An active site lysine in the protein of interest is mutated to an amber stop codon and PABK is incorporated site-specifically

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A substrate 2DPBM

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Fig. 2 (a) Rendering of PABK incorporation and decaging in the fLuc active site (PDB 4G36). (b and c) Incorporation of PABK into the fLuc529UAG-rLuc reporter. The fLuc luminescence is divided by the rLuc luminescence (fLuc/rLuc) to normalize the fLuc activity to protein produced. Error bars represent standard deviations from averages of three independent embryo lysates

by the cellular translation machinery. We have found that phosphine 2-(diphenylphosphinyl) benzamide (2DPBM, Fig. 1b) is well tolerated by zebrafish embryos and results in fast decaging of PABK and restoration of enzymatic activity [14, 15]. Thus, the phosphine can simply be added to the water of the embryos at the desired developmental time point to initiate protein function. Here we describe the protocols for expressing the caged constructs for both firefly luciferase and VP4 protease (see Note 1). Firefly luciferase contains a critical lysine in the active site (K529) essential for stabilizing the adenylated substrate intermediate during catalysis through a hydrogen bond interaction that when replaced with PABK would block substrate entry (Fig. 2a) [16]. Here, we use a dual firefly and Renilla luciferase reporter, fLucK529TAG-rLuc, that allows us to monitor the total amount of protein expressed from amber stop codon suppression (through rLuc luminescence) and also measure decaging of fLuc activity (through fLuc luminescence) [10]. Good incorporation of PABK

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into the reporter (Fig. 2b) and activation of fLuc activity through 2DPBM treatment was observed, peaking around 3 h after treatment (Fig. 2c). The viral serine protease VP4 contains a catalytic lysine in the active site (K173) that acts as a general base during catalysis, and replacement with PABK is expected to block substrate entry and disrupt its function (Fig. 3a) [17]. The protease recognizes a PXAA motif, which we inserted between a nuclear exclusion signal and mCherry to generate a fluorescent translocation reporter (NES-mCherry) to assess VP4 activation. Initial reporter characterization showed that in the absence of VP4 protease, appropriate nuclear exclusion of mCherry was observed, while co-expression of VP4 caused even dispersion of mCherry throughout the cell. As expected, mCherry remained nuclear excluded with expression of caged VP4-PABK and activation with 2DPBM affords nuclear translocation of mCherry in live embryos, similar to expression of the wild-type VP4 and reporter (Fig. 3b, c).

2

Materials

2.1 In Vitro Transcription

1. Plasmid pCS2 expressing the evolved PylRS, which encodes PABK, and the gene of interest containing an amber stop codon (TAG) at the desired incorporation site (see Note 1). 2. SP6 Transcription kit (e.g., Ambion mMessage mMachine kit). 3. T7 Transcription kit (e.g., Ambion MEGAscript kit). 4. Restriction enzyme NotI. 5. Phenol/chloroform/isoamyl alcohol mix (PCIA, 25:24:1, pH 8) (e.g., Thermo Fisher 15,593,031). 6. 3 M sodium acetate. 7. 70% and 100% ethanol. 8. Micropipettes. 9. Microcentrifuge tubes. 10. Microvolume spectrophotometer (e.g., NanoDrop).

2.2 Injection Preparation

1. 0.5% w/v phenol red solution. 2. 100 mM PABK in DMSO. 3. 35 mm and 10 cm petri dishes. 4. Mineral oil. 5. Needle puller (e.g., David Kopf Instruments, model 720). 6. Microcapillary tubes (e.g., Drummond Scientific Company, N51A custom glass tubing, 0.2 mm internal diameter, 10.6 cm length).

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Fig. 3 (a) Rendering of PABK incorporation and decaging in the VP4 active site (PDB 2GEF). (b) Micrographs of nuclear translocation assays with zoomed in regions on the right. (c) Quantification of VP4 activity by measuring the mCherry fluorescence nuclear/cytoplasmic ratio (N/C). NT = reporter only expression, WT = wild-type VP4 expression. Error bars represent standard deviations from averages of 20 cells. Scale bar = 40 μm

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2.3 Zebrafish Embryo Microinjection

1. Razor blade or scalpel. 2. Wash bottles with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) for storing and washing embryos. 3. Agarose microinjection plate: 3% agarose with 10-5% w/v methylene blue in a 10 cm petri dish with a 150-divot mold. 4. Eyelash embryo manipulator. 5. Plastic transfer pipettes. 6. Pneumatic microinjector. 7. Manual micromanipulator. 8. Dissecting stereoscope. 9. 1.7-L zebrafish slope breeding tanks. 10. Egg catcher (fine mesh strainer).

2.4 Zebrafish Embryo Luciferase Assays

1. Dual-luciferase reporter assay system. 2. Plate spectrophotometer with injector module. 3. Tabletop microcentrifuge. 4. 50 mM 2DPBM in DMSO.

2.5 Conditional Activation of a Protease with Phosphine Treatment

3

1. 1.5% low melting-point agarose in E3 media. 2. Glass-bottom 35 mm petri dish. 3. 0.0168% w/v ethyl 3-aminobenzoate (tricaine) in E3 media. 4. Scanning confocal microscope.

Methods

3.1 In Vitro Transcription of mRNA

1. Linearize ~10 μg of the pCS2 plasmid, preferably with NotI (see Note 1), in a 20 μL reaction at 37 °C for about 1 h. 2. Purify the linearized plasmid by PCIA extraction and ethanol precipitation. First, dilute the linearization reaction mixture with MilliQ water to a total volume of 50 μL and add 50 μL of PCIA. Vortex the suspension and centrifuge at 16,200 rcf for 1 min. Carefully pipette the top aqueous layer into a new microcentrifuge tube, add 5 μL of 3 M sodium acetate to the aqueous layer (1/10 volume of DNA solution), and precipitate the DNA through addition of 138 μL of 100% ethanol (2.5× volume of DNA solution) at -20 °C overnight. The next day, pellet at 16,200 rcf for 5 min. Wash the DNA pellet with 70% ethanol, pellet again through centrifugation, remove the supernatant by pipette, and dry the pellet at room temperature with the cap open for 5 min before dissolving it in 20 μL MilliQ

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water. Determine the concentration of the linearized plasmid by a microvolume spectrophotometer. 3. Use the linearized plasmid in a 20 μL SP6 in vitro transcription reaction following manufacturer instructions. For Ambion mMessage mMachine kit, set up the reaction as follows: 1 μg of linear DNA template, 10 μL of NTP/cap mix, 2 μL of 10× buffer, 2 μL of SP6 RNA polymerase, and MilliQ water to bring to a final volume of 20 μL. Incubate reaction mixtures at 37 °C for 1–2 h, followed by addition of 1 μL of Turbo DNase and incubation for 15 min at 37 °C. Purify the RNA by PCIA extraction and ethanol precipitation as described above. Generate a stock solution of RNA in 20 μL of MilliQ water and store at -80 °C. Make a 1/10 dilution of the RNA in MilliQ water for concentration measurement with a microvolume spectrophotometer. Expected concentrations of the dilution range from 400 to 1000 ng/μL. 4. Assess the quality of the RNA by agarose gel electrophoresis by loading 10 μL of the diluted solution onto a 0.8% agarose-TBE gel (40 mL) containing 0.2 μg/mL ethidium bromide and electrophorese alongside a 1-kb DNA ladder for 40 min at 80 V in TBE buffer. Image under UV light (see Note 2). 3.2 In Vitro Transcription of the PylT

1. Generate the pyrrolysine tRNA (PylT) through in vitro transcription using a T7 transcription. First, PCR amplify the template PylT (5′-taatacgactcactataggaaacctgatcatgtagatcgaacgga ctctaaatccgttcagccgggttagattcccggggtttccgcca) with a U25C mutation and CCA at the 3′ end to increase aminoacylation efficiency [10]: 34 μL of MilliQ water, 5 μL of 10× Taq buffer (KCl), 3 μL of 25 mM MgCl2, 2.5 μL of each primer (100 μM), 1 μL of 10 mM dNTP mix, 1 μL of 10 ng/μL DNA template, a nd 1 μL of 5 U/μL Taq polymerase. Use PCR primers (forwa rd: 5′-aatacgactcactatagga; reverse: 5′-tggcggaaaccccgggaa tctaa) to install a truncated T7 promotor at the 5′ end. Use the following thermocycler settings: 95 °C for 3 min; followed by 34 cycles of 95 °C for 30 s, 45 °C for 30 s, then 72 °C for 90 s; finished with 72 °C for 5 min. Purify the PCR product by PCIA extraction and ethanol precipitation as mentioned above (see Note 3). 2. For Ambion MEGAscript kit, use 1 μg of this purified PCR product in a 20 μL T7 MegaScript in vitro transcription reaction incubated at 37 °C for 5 h. At the end of the reaction, add 1 μL of TurboDNase and incubate for 15 min at 37 °C. Purify the product with PCIA extraction and then ethanol precipitation as detailed above. 3. Refold the PylT using a thermocycler: 95 °C for 1 min and then 5 °C drops every 15 s until the temperature reaches 10 °C (see

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Note 4). Make a 1/10 dilution with MilliQ water and check the RNA concentration with a microvolume spectrophotometer (see Note 5). 4. Assess the quality of the PylT by agarose gel electrophoresis. Load the rest of the diluted RNA solution onto a 40 mL 1.5% agarose-TBE gel containing 0.2 μg/mL ethidium bromide alongside a low-range DNA ladder and electrophorese for 40 min at 80 V in TBE buffer and then image under UV light. Expect a single band around 100 bp if the transcription reaction is successful. 3.3 Zebrafish Microinjections

1. Following a general protocol [18], the evening before embryo injections, set up AB* line fish by placing 4–8 fish of each sex in a segregated breeding tank (see Note 6). 2. The morning of injections, thaw the mRNA solutions for the gene of interest (i.e., containing an amber stop codon for noncanonical amino acid insertion), PylRS, and PylT on ice, as well as the PABK stock solution. PABK can be synthesized according to literature protocol [13]. Make an injection solution at a total volume of 2 μL containing the gene of interest mRNA, PylRS mRNA, PylT, and 10 mM PABK (from 100 mM stock in DMSO) (see Note 7). Add phenol red (0.1 μL, final concentration of 0.025%) to the injection solution. Carefully place a drop of the solution into the center of a 35 mm-diameter petri dish and cover it with mineral oil to prevent evaporation. This mixture should be stored on ice until injection time. 3. Use a needle puller to make 2–4 needles from microcapillary tubes. Only one is needed for each injection solution, but extra is helpful in case they break during injection setup. Using a fine-tip marker and a ruler under a dissecting stereoscope, mark each millimeter along the needle starting at the beveled edge and moving away from that point for at least 5 mm (see Note 8). 4. After preparing the injection solutions and needles, transfer the previously arranged fish to a fresh tank and remove the segregation barrier to begin mating. Plan this timing carefully so that mating begins within 2 h of the facility lights being turned on. Usually, within 30 min there will be enough embryos to begin injections; however, check embryos frequently to ensure injections can take place at the 1-cell stage for maximum production of PABK-containing protein (see Note 9). 5. In the meantime, working at a stereoscope equipped with a pneumatic microinjector, remove the very tip of the needle (2–3 mm) using a scalpel or razor blade. Be cautious not to remove too much or too little of the tip; this can make injecting the proper amount of the solution quite difficult. Insert the

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needle into the microinjector mounted on a micromanipulator (to facilitate fine movement during injections). 6. Next, collect the embryos by transferring the adult fish to a new tank and pouring the water and embryos into an egg catcher (small handheld sieve). Gently rinse the embryos into a 10 cm dish with E3 media from a wash bottle. With a transfer pipette and a plastic inoculation loop, line up embryos on an injection tray made of 3% agarose with divots to hold each embryo and then remove the excess water. The injection tray is made by pouring melted 3% agarose in E3 media supplemented with 10-5% methylene blue (to prevent fungal growth) into a 10 cm petri dish and placing a silicone zebrafish microinjection mold gently onto the surface of the agarose. Once solidified at room temperature, carefully remove the mold and wash the agarose injection tray with water. Store it at 4 °C. 7. Take the injection mix off ice and place it at the center of the stereoscope’s field of view. Carefully guide the needle tip through the mineral oil directly into the injection solution. Begin filling the needle with the solution using vacuum until several needle marks are covered. Measure the injection rate by tracking the time to inject 1 mm as marked on the needle, which in our needle’s case is approximately 30 nL. The injector can then be set to inject for a certain amount of time to achieve consistent injection of 2 nL for each embryo. Inject as soon as possible, as the RNA contents of the solution are not stable at room temperature. 8. Inject 2 nL of the injection mix into either the yolk (directly below the cell) or a single cell under a stereoscope (see Note 10) [19–21]. Once all embryos have been injected, gently wash them into a 10 cm dish with E3 media from a squirt bottle and incubate them at 28.5 °C in the dark. 3.4 Activating Luciferase with 2DPBM

1. After injection, incubate embryos expressing fLuc529PABKrLuc until 24 h post-fertilization (hpf) at 28.5 °C. At 24 hpf, transfer embryos into a 35 mm petri dish containing 2 mL of E3 media supplemented with 100 μM 2DPBM (diluted from a 50 mM DMSO stock; see Note 11). To prevent dilution of the solution, transfer embryos into a dry dish, and pipette excess water away carefully. Add the 2DPBM to 2 mL of E3 media in a 15 mL conical tube and vortex, then pour the E3 media plus 2DPBM solution into the dish, and return it to the 28.5 °C incubator. 2. After 1.5 h of incubation, replace the 2DPBM media with fresh 100 μM 2DPBM in 2 mL of E3 media and incubate for an additional 1.5 h at 28.5 °C (see Note 12).

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3. Transfer embryos into 1.7 mL microcentrifuge tubes using a plastic transfer pipette. Collect at least three tubes for each condition and include a total of four embryos per tube. Carefully remove excess media from the tube by micropipette (see Note 13). 4. Make a stock of passive lysis buffer (dilute a 5× stock included in the assay kit to 1× with DI water) and add 50 μL to each tube of embryos. Manually homogenize the embryos with the micropipette tip by pipetting up and down rapidly 20× and gently scraping the bottom and edges of the tube. 5. After homogenization, centrifuge the lysate at 16,200 rcf for 8 min at 4 °C. Meanwhile, prepare the luciferase assay components (LAR and Stop and Glow buffers) according to the manufacturer instructions and allow them to warm to room temperature. Store the ready-to-use substrate solutions as 10 mL aliquots in 15 mL conical tubes at -80 °C until needed. 6. Once the lysates finish centrifuging, collect the tubes and pipette 30 μL of lysate from each tube into a well of a whitebottomed 96-well plate (see Note 14). Take care not to introduce bubbles, and make sure the solution settles at the bottom of the well. 7. Using a plate reader with an automatic injection module, place the LAR reagent (fLuc substrate) in injector A and the Stop and Glow reagent (rLuc substrate) in injector B. Prime both injectors with 700 μL of the corresponding reagent. We use a dualluciferase assay protocol designed to dispense 20 μL of LAR into a well, wait 2 s, take a luminescence reading (automatic attenuation mode, 1-s exposure), then dispense 20 μL of Stop and Glow, wait 2 s, and take another luminescence reading (same settings). This process is repeated for each well of the 96-well plate. Conduct all assays at room temperature. 8. Load the plate into the plate reader and begin the protocol. Data should be deposited into a spreadsheet. 9. Analyze the results by first dividing the fLuc luminescence values by its corresponding rLuc luminescence values. Average triplicate samples for the rLuc and new fLuc/rLuc results and calculate standard deviations. Because fLuc luminescence can vary based on how much protein was expressed, we divided the fLuc values by the rLuc value to correct for total protein present; therefore, fLuc/rLuc measurements can be directly correlated to the decaging of PABK. The plain rLuc values represent the amount of amber stop codon suppression, allowing us to assess the efficiency of PABK incorporation.

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1. After injection (see Subheading 3.3), incubate embryos expressing VP4-PABK and the NES-mCherry reporter until 24 hpf at 28.5 °C. At 24 hpf, transfer embryos into a 35 mm petri dish containing 2 mL of E3 media supplemented with 100 μM 2DPBM and return the dish to the 28.5 °C incubator. 2. After 1.5-h incubation, replace the 2DPBM media with fresh 100 μM 2DPBM in 2 mL of E3 media and incubate an additional 1.5 h at 28.5 °C. 3. Meanwhile, make the 1.5% low melting-point agarose by mixing 15 mg of low melting-point agarose with 1 mL of E3 media in a 1.7 mL microcentrifuge tube and melt the agarose on an 80 °C heat block. 4. Make a solution of tricaine (from a 25× stock, final concentration of 0.0168% w/v in E3 media) and add about 2 mL to a 35 mm petri dish. Once 2DPBM incubations are complete, manually dechorionate the embryos using fine-nosed tweezers and transfer them to the tricaine solution. Incubate the embryos for about 10 min in this solution at room temperature for complete anesthetization. 5. Transfer the anesthetized embryos to a 35 mm glass-bottom petri dish and remove most, but not all, of the water with a micropipette. Use a separate dish for each condition and aim to image about five embryos. 6. Ensure the low melting-point agarose solution is fully melted and let it cool to about 40 °C on the benchtop at room temperature. Pipette about 400 μL of the solution on top of the embryos in the 35 mm glass-bottom petri dishes. Quickly reposition the embryos with an eyelash manipulator to be in the center of the plate and all generally in the same orientation. Repeat this for the other samples before the agarose solidifies. It can be helpful to have multiple aliquots of the 1.5% low melting-point agarose solution on the heat block. Let the agarose solidify completely at room temperature, and then add tricaine solution on top. 7. Perform embryo imaging on a scanning confocal microscope using the 555-nm laser and 20x water immersion objective using a 1024 × 1024 pixel window for imaging. Set laser power between 40% and 80% with a dwell time of 15 μs, gain at 900, and the pinhole at 1 airy unit. Acquire images in several different tissues of multiple embryos (see Note 15). 8. Analyze fluorescent micrographs with ImageJ. Using the circle selection tool, select a small region within the nucleus or cytoplasm of a cell and measure the mean fluorescence intensity. Subtract the background mean fluorescence intensity from each value and divide the nuclear value by the cytoplasm value to get the N/C ratio. Repeat this for 20 cells for each condition, average the N/C ratios and calculate standard deviations.

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Notes 1. The wild-type PylRS construct was obtained from Addgene (plasmid 99,222). To generate the PABKRS the following mutations are introduced: L274A, C313S, and Y349F [22]. It is important that linearization of the template plasmid be conducted with a restriction enzyme that leaves a 5′ overhang that cuts 3′ to the SV40 polyA sequence. Enzymes like KpnI that leave a 3′ overhang can lead to transcription of the opposite strand and result in a nonfunctional or toxic RNA product. We use NEB NotI-HF for this purpose. BSNV VP4 protease sequence: atggccgacctgcccatcagcct gctgcagaccctggcctacaagcagcccctgggcaggaacagc aggatcgtgcacttcaccgacggcgccctgttccccgtggtgg ccttcggcgacaaccacagcaccagcgagctgtacatcgccg tgaggggcgaccacagggacctgatgagccccgacgtgaggga cagctacgccctgaccggcgacgaccacaaggtgtggggcgcc acccaccacacctactacgtggagggcgcccccaagaagcccc tgaagttcaacgtgaagaccaggaccgacctgaccatcct gcccgtggccgacgtgttctggagggccgacggcagcgccgac gtggacgtggtgtggaacgacatgcccgccgtggccggccaga gcagcagcatcgccctggccctggccagcagcctgcccttcg tgcccaaggccgcctacaccggctgcctgagcggcaccaacg tgcagcccgtgcagttcggcaacctgaaggccagggccgccc acaagatcggcctgcccctggtgggcatgacccaggacggc ggcgaggacaccaggatctgcaccctggacgacgccgccgacc acgccttcgacagcatggagagcaccgtgaccaggcccgagag cgtgggccaccaggccgccttccagggctggttctactgcgg cgccggcggcggtagcggcggttctggcggtggttacccctac gacgtgcccgactacgcctga.

fLuc-hrLuc sequence:

atggaagacgccaaaaacataaagaaagg

cccggcgccattctatccgctggaagatggaaccgctggagag caactgcataaggctatgaagagatacgccctggttcctgg aacaattgcttttacagatgcacatatcgaggtggacatcac ttacgctgagtacttcgaaatgtccgttcggttggcagaagc tatgaaacgatatgggctgaatacaaatcacagaatcgtc gtatgcagtgaaaactctcttcaattctttatgccggtgtt gggcgcgttatttatcggagttgcagttgcgcccgcgaacgac atttataatgaacgtgaattgctcaacagtatgggcatttcgc agcctaccgtggtgttcgtttccaaaaaggggttgcaaaaaa ttttgaacgtgcaaaaaaagctcccaatcatccaaaaaatta ttatcatggattctaaaacggattaccagggatttcagtcga tgtacacgttcgtcacatctcatctacctcccggttttaa tgaatacgattttgtgccagagtccttcgatagggacaaga caattgcactgatcatgaactcctctggatctactggtctgcc taaaggtgtcgctctgcctcatagaactgcctgcgtgagattc

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tcgcatgccagagatcctatttttggcaatcaaatcattcc ggatactgcgattttaagtgttgttccattccatcacggtttt ggaatgtttactacactcggatatttgatatgtggatttcgag tcgtcttaatgtatagatttgaagaagagctgtttctgaggag ccttcaggattacaagattcaaagtgcgctgctggtgccaa ccctattctccttcttcgccaaaagcactctgattgacaaata cgatttatctaatttacacgaaattgcttctggtggcgct cccctctctaaggaagtcggggaagcggttgccaagaggttcc atctgccaggtatcaggcaaggatatgggctcactgagactac atcagctattctgattacacccgagggggatgataaaccggg cgcggtcggtaaagttgttccattttttgaagcgaaggttg tggatctggataccgggaaaacgctgggcgttaatcaaagagg cgaactgtgtgtgagaggtcctatgattatgtccggttatg taaacaatccggaagcgaccaacgccttgattgacaaggatgg atggctacattctggagacatagcttactgggacgaagacg aacacttcttcatcgttgaccgcctgaagtctctgattaag tacaaaggctatcaggtggctcccgctgaattggaatccat cttgctccaacaccccaacatcttcgacgcaggtgtcgcaggt cttcccgacgatgacgccggtgaacttcccgccgccgttgtt gttttggagcacggaaagacgatgacggaaaaagagatcgt ggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcg cggaggagttgtgtttgtggacgaagtaccgaaaggtcttacc ggaaaactcgacgcaagaaaaatcagagagatcctcataaagg ccaagaagggcggaaagatcgccgtgggcggcggtagcggcgg ttctggcggtggtatggcttccaaggtgtacgaccccgagcaa cgcaaacgcatgatcactgggcctcagtggtgggctcgctg caagcaaatgaacgtgctggactccttcatcaactactatg attccgagaagcacgccgagaacgccgtgatttttctgcatgg taacgctgcctccagctacctgtggaggcacgtcgtgcctcac atcgagcccgtggctagatgcatcatccctgatctgatcggaa tgggtaagtccggcaagagcgggaatggctcatatcgcctcct ggatcactacaagtacctcaccgcttggttcgagctgctgaa ccttccaaagaaaatcatctttgtgggccacgactggggggc ttgtctggcctttcactactcctacgagcaccaagacaagatc aaggccatcgtccatgctgagagtgtcgtggacgtgatcgagt cctgggacgagtggcctgacatcgaggaggatatcgccctgat caagagcgaagagggcgagaaaatggtgcttgagaataactt cttcgtcgagaccatgctcccaagcaagatcatgcggaaact ggagcctgaggagttcgctgcctacctggagccattcaaggag aagggcgaggttagacggcctaccctctcctggcctcgcgag atccctctcgttaagggaggcaagcccgacgtcgtccagattg tccgcaactacaacgcctaccttcgggccagcgacgatctg cctaagatgttcatcgagtccgaccctgggttcttttccaacg ctattgtcgagggagctaagaagttccctaacaccgagttcg tgaaggtgaagggcctccacttcagccaggaggacgctccag atgaaatgggtaagtacatcaagagcttcgtggagcgcgtg ctgaagaacgagcagtaa,

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2. This is not a denaturing gel, so the mRNA secondary structure can lead to multiple bands. In our experience however, there are usually two bands, one at the expected molecular weight (using a DNA ladder) and one that can be higher or lower on the gel. Smearing may indicate degradation of the RNA (or overloading the gel). Absence of bands usually indicates failure of in vitro transcription. Even if the measured RNA concentration is high, the reaction could still have failed and the collected product may actually be short, failed transcripts that are not visible on the gel. 3. It is especially important to use PCIA extraction and ethanol precipitation here, as the PCR product is too small for column purification. 4. The melting and refolding help keep the PylT soluble at high concentrations. 5. We would usually combine two 20 μL in vitro transcription reactions after PCIA extraction and before ethanol precipitation to increase the concentration of the final PylT aliquot. In our experience, a stock solution between 10 and 20 μg/μL of PylT is easiest to use for the generation of injection solutions that allow for good incorporation of PABK into protein. 6. We use 1.7-L beach tanks which promote egg release by the females and resulted in reliable embryo collection every week. In our experience, having about a 2:1 female/male ratio as well as the inclusion of a small plastic plant yields the most fertilized embryos. Adjust the number of total fish based on the tank size. 7. Our standard injection solution recipe for noncanonical amino acid incorporation in zebrafish embryos is 200 ng/μL of mRNA corresponding to the target protein with an amber stop codon at the site for noncanonical amino acid, 200 ng/μ L of PylRS mRNA, 5–10 μg/μL of PylT, and 10 mM of the noncanonical amino acid. If the target protein is toxic, less mRNA can be added. 8. We use a specific type of microcapillary tube with a calibrated volume of 30 nL for every mm length. This is used to calculate the time needed to inject 1–2 nL/embryo. Of course, this calculation may change for other brands of microcapillary tubes. An alternative way to measure injection rate is through measurement of the bubble diameter produced in mineral oil. Additionally, the needle puller parameters will need to be optimized for each type of microcapillary tube used. 9. Zebrafish are most actively breeding soon after the light cycle begins. It is still possible to collect embryos later in the morning, but we have had the best success with starting breeding around 1 h after the lights come on in the fish facility. For

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homogeneous expression of injected mRNA, injections at the one-cell stage of embryo development (first 45–60 min postfertilization at room temperature) are recommended. However, we have been successful in homogenous expression after injecting in the yolk of 2–8 cell embryos due to injection contents being dispersed to all cells by cytoplasmic streaming [19–21]. 10. For all noncanonical amino acid incorporations performed in zebrafish, we observe no difference in the amount of protein produced injecting into the cell or the yolk. The yolk is much larger and easier to penetrate with a needle, so all results shown here are from injections into the yolk. However, although not used in this chapter, injection of plasmid DNA requires direct cell injection [23, 24]. 11. We have tested several other phosphines and found 2DPBM had the fastest decaging of an aryl azide-caged rhodamine sensor in zebrafish embryos [14]. It is also well tolerated in zebrafish embryos with no toxicity or deformity seen with 3-h incubations. 12. The half-life of oxidation of 2DPBM in E3 media was found to be 90 min [14]; hence, we replace 2DPBM solutions at 90 min. A total of 3-h incubation should be sufficient for full activation of fLuc. 13. More embryos can be collected per tube if protein expression is low. In general, four embryos are sufficient for luciferase assays. Once the excess water is removed, it is possible to store the embryos at -80 °C until the assay is performed. 14. The white-bottom plates result in higher luminescence values and greater sensitivity than clear-bottom plates. Also of note, if running any wild-type luciferase samples, these should be at opposite ends of the plate or preferably at the end of the run or on a separate plate, as very high luminescence can bleed into measurements of proximal wells. 15. We have found the 24-hpf embryo brain and tail muscle to have excellent expression of the mCherry reporter for analysis of N/C ratios. The brain is also very cell-dense, allowing for collection of many measurements from a single image.

Acknowledgements We acknowledge financial support from the National Institutes of Health (R01GM132565). W.B. was supported by a University of Pittsburgh Mellon Fellowship. Parts of figures were generated using BioRender.com.

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References 1. Meyers JR (2018) Zebrafish: development of a vertebrate model organism. Curr Protoc Essent Lab Tech 16(1):e19. https://doi.org/ 10.1002/cpet.19 2. Teame T, Zhang Z, Ran C, Zhang H, Yang Y, Ding Q et al (2019) The use of zebrafish (Danio rerio) as biomedical models. Anim Front 9(3):68–77. https://doi.org/10.1093/ af/vfz020 3. Bradford YM, Toro S, Ramachandran S, Ruzicka L, Howe DG, Eagle A et al (2017) Zebrafish models of human disease: gaining insight into human disease at ZFIN. ILAR J 58(1):4–16. https://doi.org/10.1093/ilar/ ilw040 4. Kalvaityte˙ M, Balciunas D (2022) Conditional mutagenesis strategies in zebrafish. Trends Genet 38(8):856–868. https://doi.org/10. 1016/j.tig.2022.04.007 5. Varady A, Distel M (2020) Non-neuromodulatory optogenetic tools in zebrafish. Front Cell Dev Biol 8:418. https:// doi.org/10.3389/fcell.2020.00418 6. Antinucci P, Dumitrescu A, Deleuze C, Morley HJ, Leung K, Hagley T et al (2020) A calibrated optogenetic toolbox of stable zebrafish opsin lines. elife 9:e54937. https://doi.org/ 10.7554/eLife.54937 7. Deiters A, Yoder JA (2006) Conditional transgene and gene targeting methodologies in zebrafish. Zebrafish 3(4):415–429. https://doi. org/10.1089/zeb.2006.3.415 8. Pearce S, Tucker CL (2021) Dual systems for enhancing control of protein activity through induced dimerization approaches. Adv Biol 5(5):2000234. https://doi.org/10.1002/ adbi.202000234 9. Brown W, Liu J, Deiters A (2018) Genetic code expansion in animals. ACS Chem Biol 13(9):2375–2386. https://doi.org/10.1021/ acschembio.8b00520 10. Liu J, Hemphill J, Samanta S, Tsang M, Deiters A (2017) Genetic code expansion in zebrafish embryos and its application to optical control of cell signaling. J Am Chem Soc 139(27): 9100–9103. https://doi.org/10.1021/jacs. 7b02145 11. Brown W, Wesalo J, Tsang M, Deiters A (2023) Engineering small molecule switches of protein function in zebrafish embryos. J Am Chem Soc 145(4):2395–2403. https://doi.org/10. 1021/jacs.2c11366

12. Luo J, Liu Q, Morihiro K, Deiters A (2016) Small-molecule control of protein function through staudinger reduction. Nat Chem 8(11):1027–1034. https://doi.org/10.1038/ nchem.2573 13. Wesalo JS, Luo J, Morihiro K, Liu J, Deiters A (2020) Phosphine-activated lysine analogues for fast chemical control of protein subcellular localization and protein SUMOylation. Chembiochem 21(1–2):141–148. https://doi.org/ 10.1002/cbic.201900464 14. Darrah K, Wesalo J, Lukasak B, Tsang M, Chen JK, Deiters A (2021) Small molecule control of morpholino antisense oligonucleotide function through staudinger reduction. J Am Chem Soc 143(44):18665–18671. https://doi.org/10. 1021/jacs.1c08723 15. Lukasak B, Morihiro K, Deiters A (2019) Aryl azides as phosphine-activated switches for small molecule function. Sci Rep 9(1):1470. https:// doi.org/10.1038/s41598-018-37023-6 16. Jazayeri FS, Amininasab M, Hosseinkhani S (2017) Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase. PLoS One 12(7):e0180667. https://doi.org/10.1371/journal.pone. 0180667 17. Da Costa B, Soignier S, Chevalier C, Henry C, Thory C, Huet J-C et al (2003) Blotched snakehead virus is a new aquatic birnavirus that is slightly more related to avibirnavirus than to aquabirnavirus. J Virol 77(1):719–725. https://doi.org/10.1128/jvi.77.1.719-725. 2003 18. Brown W, Deiters A (2019) Chapter 13: Lightactivation of Cre recombinase in zebrafish embryos through genetic code expansion. In: Deiters A (ed) Methods in enzymology. Academic Press 77:265–281. https://doi.org/10. 1016/bs.mie.2019.04.004 19. Hammerschmidt M, Bitgood MJ, McMahon AP (1996) Protein kinase a is a common negative regulator of hedgehog signaling in the vertebrate embryo. Genes Dev 10(6): 647–658. https://doi.org/10.1101/gad.10. 6.647 20. Rosen JN, Sweeney MF, Mably JD (2009) Microinjection of zebrafish embryos to analyze gene function. J Vis Exp 25:e1115. https:// doi.org/10.3791/1115 21. Culp P, Nu¨sslein-Volhard C, Hopkins N (1991) High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish

Conditional Protein Control in Zebrafish Embryos eggs. Proc Natl Acad Sci 88(18):7953–7957. https://doi.org/10.1073/pnas.88.18.7953 22. Ge Y, Fan X, Chen PR (2016) A genetically encoded multifunctional unnatural amino acid for versatile protein manipulations in living cells. Chem Sci 7(12):7055–7060. https:// doi.org/10.1039/C6SC02615J 23. Mumm JS, Williams PR, Godinho L, Koerber A, Pittman AJ, Roeser T et al (2006)

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Chapter 19 Noncanonical Amino Acid Incorporation in Mice Zhetao Zheng and Qing Xia Abstract Genetic code expansion enables in cellulo biosynthesis of curative proteins with enhanced specificity, improved stability, and even novel functions, due to the incorporation of artificial, designed, noncanonical amino acids (ncAAs). In addition, this orthogonal system also holds great potential for in vivo suppressing nonsense mutations during protein translation, providing an alternative strategy for alleviating inherited diseases caused by premature termination codons (PTCs). Here we describe the approach to explore the therapeutic efficacy and long-term safety of this strategy in transgenic mdx mice with stably expanded genetic codes. Theoretically, this method is applicable to about 11% of monogenic diseases involving nonsense mutations. Key words Genetic code expansion, Non-canonical amino acids, Gene targeting, Nonsense mutation diseases, Translation machinery

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Introduction Expanding genetic code using the orthogonal translation elements, which consists of an engineered aminoacyl-tRNA-synthase (aaRS)– tRNA pair and relevant noncanonical amino acids (ncAAs), enables precise manipulation of protein structures and functions in bacterial and mammalian cells by site-specific incorporation of ncAAs into proteins [1, 2]. Among established aaRS–tRNA pairs, the PylRS– tRNA pair and its derivatives are most widely used for decoding nonsense codons (i.e., UAG, UAA, and UGA) within exogenous genes into ncAAs in mammalian cells, so as to produce engineered proteins for research and therapy [3, 4]. Using viral vector delivery of PylRS–tRNA pairs and injection of ncAAs, time-resolved protein labeling and spatial proteomics studies depending on protein modification could be accomplished in the mouse brain, which lay the foundation for genetic code expansion in vivo [5, 6]. However, utilization of this system to read-through endogenous premature termination codons are barely exploited to date.

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2_19, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Premature termination codons in human genes result in nonsense mutation diseases, which account for approximately 11% of reported monogenic diseases [7, 8]. For example, more than 5% of Duchenne muscular dystrophy (DMD), a rare human Xchromosome-linked muscular disease, are caused by truncated dystrophin proteins produced from the DMD gene harboring nonsense mutations [9, 10]. Previously, we have engineered the PylRS– tRNA pair to specifically recognize and charge a desired ncAA (Nε-2-azidoethyloxycarbonyl-L-lysine, referred as NAEK) in response to the UAA nonsense codon in DMD transcripts. Moreover, we have demonstrated the potential of aaRS–tRNA pairs to restore endogenous dystrophin expression and reduce symptoms by nonsense read-through in vitro and in vivo, suggesting a feasible approach to treat nonsense mutation diseases [11]. In this chapter, we provide detailed instructions to generate PylRS – tRNAPyl UUA - GFP39TAA transgenic mice with CRISPR–Cas9 gene editing technology [12] as a reliable animal model for evaluating the long-term safety of the PylRS–tRNA system and subsequently investigate its therapeutic potential for DMD by crossing the transgenic mice with mdx mice.

2

Materials Prepare all solutions using Ultrapure water (i.e., purified deionized water with a resistivity >18 MΩ-cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.

2.1 Molecular Cloning and Protein Immunoblotting

1. Lysogeny broth (LB) medium: Dissolve 8 g NaCl, 8 g tryptone, and 4 g yeast extract in 800 mL RNase-free water. Sterilize by autoclaving for 20 min at 15 psi and 121 °C. Store at 4 ° C for no more than 2 months. 2. LB agar: Dissolve 8 g NaCl, 8 g tryptone, 4 g yeast extract, and 12 g agar in 800 mL RNase-free water. Sterilize by autoclaving for 20 min at 15 psi and 121 °C. Cool down to about 60 °C on ice, and then add 800 μL of 100 mg/mL ampicillin. Pour 15 mL of the LB agar solution into each petri dish. Allow the solution to solidify in room temperature and store at 4 °C. 3. Lysis buffer: 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 1 mg/mL lysozyme, and 100 μM phenylmethylsulphonyl fluoride (PMSF). 4. 4 × SDS-PAGE loading dye: 200 mM Tris–HCl pH 6.8, 400 mM dithiothreitol (DTT), 8% sodium dodecyl sulphate (SDS), 0.4% bromophenol blue, and 40% glycerol.

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5. SDS-PAGE running buffer: 0.025 M Tris–HCl, pH 8.3, 0.192 M glycine, and 0.1% SDS. 6. Western blot transfer buffer: 0.025 M Tris–HCl, 0.192 M glycine, and 20% methanol. 7. Phosphate-buffered saline (PBS): 11.4 mM NaH2PO4, 38.6 mM NaH2PO4, 150 mM NaCl, pH 7.4. 8. Tris-buffered Tween saline (TBST) buffer: 50 mM Tris–HCl, 150 mM NaCl, and 0.02% Tween-20, pH 7.5. 9. Blocking solution for western blotting: 5% milk in TBST. Store at 4 °C. 10. PBST buffer: PBS with 0.1% Triton X-100. 11. Blocking buffer for immunostaining: 5% normal donkey serum in PBST. Store at 4 °C. 12. Plasmid pCS vector (Addgene plasmid # 12158). 13. Microvolume spectrophotometer for determining DNA concentration. 14. Setup for PCR and DNA gel electrophoresis. 15. Protein quantification kit (e.g., BCA assay). 16. NuPAGE gels and protein electrophoresis apparatus. 17. Antibodies: goat anti-rabbit IgG Alexa Fluor 594 (1:400, A-11037, Thermo Fisher), anti-dystrophin antibody (1:100, ab15277, Abcam), HRP-conjugated goat anti-rabbit IgG (1: 3000, bs-0294R-HRP, Bioss), anti-GFP antibodies (1:3000, 66002-1-Ig, Proteintech), and anti-GAPDH antibodies (1: 3000, sc-365062, Santa Cruz Biotechnology). 18. 4% paraformaldehyde (PFA). 19. 0.5 μg/mL Hoechst. 2.2

Cell Culture

1. HEK293T cells. 2. Cell culture medium: DMEM containing 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin. Store at 4 °C. 3. Cell freezing medium: fetal bovine serum containing 10% DMSO. Ready to use. 4. Transfection reagent.

2.3

Animal

1. C57BL/6 mice. 2. 1.25% aphrodine solution (w/v) for anesthesia: Dissolve 5 g aphrodine in 10 mL tert-amyl alcohol to a concentration of 0.42 g/mL. Store this brown color solution at 4 °C. For use, dilute the storage solution with saline (300 μL of storage solution in 100 mL of saline) to the working concentration. 3. 70% alcohol.

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4. Alcohol lamp. 5. Human chorionic gonadotrophin (HCG). 6. 500 U/mL hyaluronidase. 7. Microscope. 8. 100 mg/mL NAEK solution: Dissolve 1 g NAEK powder in 10 mL of PBS or saline in a 15 mL centrifuge tube, following sterilization by a 0.2 μm membrane filter and then storing at 4 ° C (see Note 1). 2.4

Quantification

1. Dark box for western blotting (e.g., Fuji Las-3000). 2. Quantification software: ImagePro Plus (version 6).

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Methods The step-by-step method details could be divided into two main parts. The first is to generate transgenic mice by embryo microinjection and transfer [13], and the second is to systematically analyze and evaluate the in vivo efficacy and safety of the PylRS–tRNA system for DMD therapy. Carry out all procedures at room temperature unless otherwise specified. All animal experiments need to be performed with permission of the relevant authority/ institution. In this part, we focus on describing the design and construction of the targeting vector and donor template for genetic integration of PylRS – tRNAPyl UUA - GFP39TAA transgene in mice (Fig. 1). Preparation of fertilized eggs for microinjection, which is adapted from the previous methods [14, 15], is also included.

3.1 Plasmid Construction and Linearization

Generate a plasmid for expressing CRISPR/Cas9 and relevant single-guide RNA (sgRNA) in vivo, enabling efficient knock-in of the gene cassettes carrying PylRS – tRNAPyl UUA - GFP39TAA by homology-directed repair in the presence of the donor template. 1. Design sgRNAs based on the target gene sequence. In this method, we choose ROSA26, a well-characterized safe harbor locus [16], for stable integration of the transgene. Recommended sgRNAs are listed in Table 1. 2. Ligate each sgRNA to the T7 promoter, and clone into the pCS (puro) vector with CAG promoter-driven Streptococcus pyogenes (SpCas9) via Gibson assembly, respectively, and then construct the CAG-Cas9-T7-sgRNA-pCS targeting plasmid. 3. Generate the donor template by ligating the CAG promoter with MmPylRS (Table 2), IRES with GFP39TAA, together with the tRNA expression cassette consisting of a 7sk promoter and a tRNAPyl UUA. We recommend constructing at least four

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sgRNA

Cas 9 Gene 1

Gene 2

Wild ROSA26 allele

Insert

Insert

Pyl

Targeting vector (PyIRS/tRNA UUA & GFP) 5’

CAG Pro

PyIRS-IRES-EGFP 39TAA

WPRE

PA

Pyl

(7sk pro-tRNA UUA ) 4

LR (1812bp)

3’ RR (1779bp)

Fig. 1 Gene targeting for integrating PylRS – tRNAPyl UUA - GFP39TAA transgene in ROSA26 allele of mice

Table 1 Sequences of sgRNAs for targeting ROSA26 site sgRNAs

Spacer sequence (5′-3′)

g1

ACTGGAGTTGCAGATCACGAGGG

g2

GGCAGGCTTAAAGGCTAACCTGG

g3

GTCCTGCAGGGGAATTGAACAGG

g4

AAGATGGGCGGGAGTCTTCTGGG

g5

GTGTGTGGGCGTTGTCCTGCAGG

g6

TGGGCGGGAGTCTTCTGGGCAGG

g7

CGCCCATCTTCTAGAAAGACTGG

g8

CAGGACAACGCCCACACACCAGG

g9

GTTGCAGATCACGAGGGAAGAGG

g10

TCTGAGGACCGCCCTGGGCCTGG

g11

CAGGGCGGTCCTCAGAAGCCAGG

g12

AAGGCCGCACCCTTCTCCGGAGG

g13

ACCCTTCTCCGGAGGGGGGAGGG

g14

GAGCTGCAGTGGAGTAGGCGGGG

g15

GCTCTGAGTTGTTATCAGTAAGG

g16

CCTCGATGGAAAATACTCCGAGG

g17

TGGGAGGATAGGTAGTCATCTGG

g18

CGACAAAACCGAAAATCTGTGGG

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Table 2 DNA sequences of aaRS–tRNA pairs Sequences (5′-3′) MmPylRS

atggagcaaaagctcatttctgaagaggacttggataaaaaaccactaaacactctgatatctgcaaccgggctctggatgtc caggaccggaacaattcataaaataaaacaccacgaagtctctcgaagcaaaatctatattgaaatggcatgcggagac caccttgttgtaaacaactccaggagcagcaggactgcaagagcgctcaggcaccacaaatacaggaagacctgcaaa cgctgcagggtttcggatgaggatctcaataagttcctcacaaaggcaaacgaagaccagacaagcgtaaaagtcaagg tcgtttctgcccctaccagaacgaaaaaggcaatgccaaaatccgttgcgagagccccgaaacctcttgagaatacagaa gcggcacaggctcaaccttctggatctaaattttcacctgcgataccggtttccacccaagagtcagtttctgtcccggcat ctgtttcaacatcaatatcaagcatttctacaggagcaactgcatccgcactggtaaaagggaatacgaaccccattacatcc atgtctgcccctgttcaggcaagtgcccccgcacttacgaagagccagactgacaggcttgaagtcctgttaaacccaaaag atgagatttccctgaattccggcaagcctttcagggagcttgagtccgaattgctctctcgcagaaaaaaagacctgcagca gatctacgcggaagaaagggagaattatctggggaaactcgagcgtgaaattaccaggttctttgtggacaggggttttct ggaaataaaatccccgatcctgatccctcttgagtatatcgaaaggatgggcattgataatgataccgaactttcaaaacagat cttcagggttgacaagaacttctgcctgagacccatgcttgctccaaacctttacaactacctgcgcaagcttgacagggccc tgcctgatccaataaaaatttttgaaataggcccatgctacagaaaagagtccgacggcaaagaacacctcgaagagtttacca tgctgaacttctgccagatgggatcgggatgcacacgggaaaatcttgaaagcataattacggacttcctgaaccacctggga attgatttcaagatcgtaggcgattcctgcatggtctatggggatacccttgatgtaatgcacggagacctggaactttcctct gcagtagtcggacccataccgcttgaccgggaatggggtattgataaaccctggataggggcaggtttcgggctcgaacgc cttctaaaggttaaacacgactttaaaaatatcaagagagctgcaaggtccgagtcttactataacgggatttctaccaacctgta

tRNAPyl UUA

ggaaacctgatcatgtagatcgaatggactTTAaatc cgttcagccgggttagattcccggggtttccgcca

copies of the tRNA cassette (see Note 2) and obtain the donor plasmid of CAG-MmPylRS-IRES-GFP39TAA-(7sk-tRNAPyl UUA)4. 4. In vitro evaluate the editing efficiency by transfecting each targeting plasmid with the donor plasmid into HEK293T cells and measuring the knock-in efficiency. Select the sgRNA that exhibits the highest activity to perform genetic integration in vivo. 5. Treat the targeting plasmid and the donor plasmid with restriction enzymes according to the manufacturer’s protocol, and then obtain the linearized fragments after DNA extraction. Measure the concentration of each linearized fragment by the microvolume spectrophotometer and store them at -20 °C (see Note 3). 3.2 Vasectomized Male Mice

Vasectomize males to engender pseudopregnancy in mature female mice. Such pseudopregnant females act similar to the pregnant hormonally and physiologically, which are usually considered to be competent recipients for microinjected fertilized one-cell eggs. 1. Anesthetize the C57BL/6 mice by injecting with the 1.25% aphrodine solution intraperitoneally at a ratio of used solution volume to mouse body weight around 15–7 μL/g, and then place mice on a mouse surgical blanket to finish anesthesia.

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Fig. 2 Vasectomy in mice

2. Fix the anesthetized male mouse in a supine position with the abdomen upward. Use 70% alcohol to disinfect the lower abdomen and trim mouse hairs in the surgical site. 3. Make the incision in the middle of the lower abdomen between the two hind limbs, either longitudinally or transversely, at a length of approximately 1 cm, and open the skin and muscles gently. 4. Gently pull out the testes, epididymis, and vas deferens with blunt-tipped forceps. Note that the vas deferens is rich in vessels which distribute longitudinally. After the vas deferens is freed from the surrounding tissues with scissors or forceps, burn the forceps on the flame of an alcohol lamp until it turns red, and then clamp directly on the vas deferens (Fig. 2). 5. Return all organs to the abdominal cavity with blunt-tipped forceps. Repeat the above step 4 to sever the vas deferens on the other side. 6. Suture the incision and place each mouse in a single cage. Vasectomized male mice should be kept warm by light bulbs until recovery, usually in 2–3 weeks.

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7. Mate the vasectomized male mice with healthy 8-week-old female mice in estrus and observe for pregnancy to test the effectiveness of the vasectomy. 3.3 Preparing Female Mice of Pregnancy and Pseudopregnancy

1. Close the vaginal orifices of 8-week-old C57BL/6 female mice and give each mouse with 5 IU (international unit) of pregnant mare’s serum gonadotropin (PMSG) via intraperitoneal injection. 2. Divide them randomly into at least two groups for generating pregnant (donors) and pseudopregnant (recipients) mice, respectively. 3. Within 46–48 h, each mouse in the pregnant group is injected intraperitoneally with 5 IU of HCG and mated with a normal 8-week-old male in one cage. 4. Select female mice with flushed vaginal orifices (estrus) in the pseudopregnant group as recipients and mate them with two normal 8-week-old males in one cage, respectively. All mice should be reared in-house (temperature, 20–25 °C; humidity, 40–70%) under specific-pathogen-free conditions with lighting controlled (light: 08:00–20:00).

3.4 Taking Fertilized Egg

1. Find donors and recipient with sperm bolus in the next day following mating mice, and remove them from the cage. Execute donor mice while placing recipient mice in a new cage, waiting for surrogacy. 2. For the donor mice, wet their backs with 70% alcohol, pick up the skin with forceps at the back against the caudal end, cut out a transverse incision with scissors, hold the skin at the incision with forceps and peel it away towards the head side to expose the muscles of the dorsal lumbar region, make a small incision here with small scissors, and cut off the ovaries with small scissors (taking care not to cut into the fallopian tubes) (Fig. 3). 3. Cut open the uterine horns and place the severed fallopian tubes into the prepared flushing holding medium (FHM) droplet. The entire oviduct is flushed under a body microscope using an aspirated FHM syringe with a blunt-mouthed needle and inserted into the umbilical opening of the oviduct (which can be blown out with an egg transfer needle). 4. After all eggs are removed, the eggs are transferred to an approximately 200 μL droplet, 10–15 μL of 500 U/mL hyaluronidase is added to digest the granulosa cells, and after approximately 30 s, blow the droplet several times with a 200 μL pipette until all eggs are dislodged. 5. Transfer the fertilized eggs into a new FHM droplet three times using a glass pipette (120–250 μm in diameter, i.e., larger

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Fig. 3 Fertilized egg collection

than the diameter of one fertilized egg but smaller than the diameter of two fertilized eggs) to wash away the granulocytes, residue, and hyaluronidase. 6. Transfer the washed fertilized eggs to KSOM-AA medium (Millipore) droplets covered with mineral oil (which had been equilibrated overnight in a 5% CO2, 37 °C incubator to ensure the correct pH and osmotic pressure) and incubate in a 5% CO2, 37 °C incubator for a period of time. 7. Observe fertilized eggs under the microscope and select eggs with full cells, clear zona pellucida, and clearly visible polar bodies for use. 3.5 Microinjection of Linearized Targeting Vectors and Donor Template

1. Preparation of egg holders: load a 1.0 mm-diameter coreless glass tube into a needle puller and run the needle pulling program, to produce a fracture of approximately 100–120 μm in diameter. The fractured needle is then loaded into the forging needle apparatus and the fracture is melted to produce an inner diameter of approximately 15 μm, which is then placed into a homemade needle box and set aside. 2. Preparation of injection capillaries: load the 1.0 mm-diameter cored glass tube into the needle puller and run the needle pulling program, and then put the accomplished capillaries into the homemade needle box.

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3. Make a FHM droplet of about 2 mm in diameter in the center of a 6 cm petri dish, covered with mineral oil to prevent evaporation. 4. Insert the injection capillary upside down into the linearized DNA solution at a concentration of 1–3 μg/mL, and be careful to suck the DNA solution into the tip of the capillary, without any air bubbles stuck in the middle of the tube. 5. Transfer the fertilized eggs cultured in KSOM-AA into the prepared FHM droplet, place the dish on the carrier table of the inverted microscope, and load the egg holder and injection capillary into the operation arm. 6. Use a 10× objective lens and focus on the eggs until the eggs are clearly imaged. The holder and injection capillary are pulled into the FHM droplet and placed in the center, parallel to the X-axis in the field of view. 7. Run the Eppendorf syringe to create a constant pressure in the injection capillary. The objective lens is adjusted to 40× so that the nucleus can be clearly seen. Bump the injection capillary over the holder to produce a tiny break for inserting eggs. 8. Hold an egg and adjust the position of the capillary in the Z-axis direction so that the needle tip is clearly located in the same focal plane with the nucleus of egg. Blow out the linearized DNA into the egg and quickly withdraw the needle after seeing the nucleus visibly distended (Fig. 4). 9. Use the holder to push the egg down gently and aspirate a new egg for injection and so on until all eggs are injected. 3.6

Embryo Transfer

1. Anesthetize the recipient mice by injecting 1.25% aphrodine intraperitoneally. Trim hairs and disinfect skin with 70% alcohol. 2. Cut the skin and abdominal wall longitudinally at the near midline of the back. Make a small incision above the white fat body to cut the peritoneum. Hold the fat body with blunt forceps. Pull ovaries, fallopian tubes, and uterus out from the incision in succession, and then carefully move them into the body microscope. 3. Make a hard glass capillary of approximately 150–200 μm diameter, and inhale the FHM, a small bubble, the FHM, a small bubble into the capillary in order. At the end of the capillary, inhale 10–15 embryos developed to the 2-cell stage with the culture FHM (Fig. 5a). 4. Load the transfer capillary with embryos into the operation arm of the stereo microscope without vibrating and touching it. Gently tear the bladder membrane to find the oviduct and ampulla under the microscope.

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Uninjected zygotes Site of injection

Injection capillary

Holder

M2 + medium Oil

Injected zygotes

Holder Injection capillary 35º

Fig. 4 The schematic illustration of microinjection in fertilized eggs

5. Use the ophthalmic forceps to hold the fallopian tube, and then insert the capillary thereinto and expel embryos towards the ampulla. Two or more bubbles can be observed in the ampulla, indicating successful transfer (Fig. 5). 6. Withdraw the uterine and other organs back into the abdominal cavity and suture the muscle and skin, respectively. Disinfect the skin using 70% alcohol again. 7. Each mouse after transplantation is put back into a single cage equipped with cottons for keeping warm. Monitor the behaviors of surrogate mice every day and provide foods with adequate nutrition for females to breed their offspring. 3.7 Genotype Identification of Transgenic Mice by PCR Analysis

1. Reproduction of transgenic mice: mate healthy 8-week-old male transgenic mice with female normal mice in a 1:2 ratio, while female transgenic mice with normal male mice in a 2:1 ratio. When the female mice are pregnant, place them in a separate cage to breed the offspring, respectively. 2. When the offspring mice are about 3 weeks old, tag the mice with an ear punch, and then clip the tail tip to a length of 2 cm and immediately place it in a 1.5 mL centrifuge tube placed on ice. 3. Add 200 μL of lysis buffer and 20 μL of protease to each tube and place in a 55 °C water bath, shaking overnight to fully dissolve the tissue (see Note 4). 4. Centrifuge at 12,000 rcf for 10 min, remove the supernatant to a new centrifuge tube, add 200 μL of ethanol per tube, and mix thoroughly. Centrifuge at 12,000 rcf for 2 min, take the

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Fig. 5 Embryo transfer. (a) To inhale 10–15 embryos with small bubbles and the FHM in a capillary. (b) Schematic diagram of mouse reproductive organ anatomy. (c–f) Isolate of mouse ovaries, fallopian tubes, and uterus (d, e) and inject zygotes (f)

supernatant and add to the DNA adsorption column, centrifuge again, and discard the waste liquid in the collection tube. 5. Add 600 μL of wash buffer 1 to each tube, centrifuge at 12,000 rcf for 2 min, and discard the waste liquid in the collection tube. 6. Add 600 μL of wash buffer 2 per tube, centrifuge at 12,000 rcf for 2 min, and discard the waste in the collection tube. 7. Spin the adsorption tube at 12,000 rcf for 2 min and discard the waste liquid in the collection tube. 8. Replace the collection tube with a clean centrifuge tube, and add 50 μL of ddH2O (pre-warmed at 60 °C,) to each adsorption column. One minute later, centrifuge at 12,000 rcf for 2 min and collect the centrifuge tube. 9. Use a microvolume spectrophotometer to determine the concentration and purity of the extracted mouse tail DNA (see Note 5).

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Table 3 PCR primers used in amplifying transgenes of mouse tail DNA Primers

Sequences (5′-3′)

Forward-1

CGTGTTCGTGCAAGTTGAGTCCATC

Reverse-1

TTTGATAAGGCTGCAGAAGGAGCGG

Forward-2

CTACAGCTCCTGGGCAACGTG

Reverse-2

CAGCGTTTGCAGGTCTTCCTGTAT

Table 4 PCR reaction for amplifying transgenes of mouse tail DNA PCR reagents

Amount

DNA template (20–100 ng)/μL

1.0 μL

Phanta master mix

10 μL

Primer forward (10 μM)

1.0 μL

Primer reverse (10 μM)

1.0 μL

ddH2O

7.0 μL

Table 5 PCR program for amplifying transgenes of mouse tail DNA Step

Cycles

Temperature (°C)

Time

Initial denaturation

1

95

5 min

95

30 s

55

30 s

72

1 min

Denaturation Annealing

35

Extension Final extension

1

72

10 min

Hold

1

4

1

10. Design primers for PCR analysis of mouse tail DNA (Table 3) and set up reactions on ice (Table 4) and run the PCR program (Table 5) (see Note 6). 11. The genotype of each sample is determined based on the size of two PCR products by gel electrophoresis. The two primer sets, set-1 (forward-1 and reverse-1) targeting the sequence at 2773 bp and set-2 (forward-2 and reverse-2) targeting the sequence at 3384 bp. The positive transgenic mice annotated

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Fig. 6 PCR analysis of mouse tail DNA of transgenic mice. The positive transgenic mice were annotated in the figure, which was characterized by both positive bands of 2773 bp and 3384 bp

in Fig. 6 are characterized by both positive bands of 2773 bp and 3384 bp. 12. Select the positive transgenic mice for reproduction or further investigation. In Subheadings 3.8 and 3.9, we further demonstrate the noncanonical amino acid-controlled protein expression in transgenic mice, laying the foundation for protein engineering or manipulation in vivo by genetic code expansion. Moreover, we generate the trans/mdx mice by mating mdx mice with aforementioned transgenic mice and evaluate the dystrophin restoration as a proof-ofprinciple investigation in DMD therapy. 3.8 Noncanonical Amino AcidDependent Fluorescent Protein Expression in Transgenic Mice

1. Inject the mouse intraperitoneally with 500 μL of 100 mg/mL NAEK solution or 0.9% NaCl solution every 2 days. Remember to vary the side injected between right and left for multiple administrations (see Note 7). 2. Daily monitor and record the body weight and survival rate of the transgenic mice injected with NAEK or 0.9% NaCl solution for safety evaluation (Fig. 7). 3. Detection of GFP39TAA expression in different tissues of transgenic mice via western blot. Harvest heart, muscle, liver, and kidney tissues of the transgenic mice injected intraperitoneally with NAEK or 0.9% NaCl solution. 4. Add an appropriate amount of lysis buffer and protease inhibitor for full lysis, and centrifuge at 16,000 rcf, 4 °C, for 15 min to collect the supernatants. 5. Quantify protein concentration, and then boil 100 μg of protein from each sample with 4 × SDS-PAGE loading dye.

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Fig. 7 Body weight curves (a) and survival rates (b) of different mice used in this method for evaluating the long-term safety of PylRS–tRNA system

6. Perform electrophoresis on 4–12% NuPAGE with a voltage of 120 V. 7. Before use, activate a polyvinylidene difluoride membrane with methanol for 30 s, and do remove air bubbles between the layers with a glass rod. 8. Electroblotting in a 4 °C refrigerator with a voltage of 100 V for 1 h (see Note 8). 9. Block the membrane with 5% (v/v) nonfat milk in TBST at room temperature for 1 h. 10. Incubate the membrane with anti-GFP antibodies and antiGAPDH antibodies diluted in TBST containing 5% (v/v) of defatted milk, overnight at 4 °C. 11. Rinse the incubated membrane with TBST at 130 rpm for 10 min, and repeat for three times. 12. Incubate the membrane with HRP-conjugated IgG at room temperature for 1 h. 13. Rinse the incubated membrane with TBST for three times. 14. Apply the luminescent solution evenly to the membrane. 15. The protein bands are visualized in a Fuji Las-3000 dark box (FujiFilm), and the integrated optical density (IOD) of western blot bands is semiquantified using the ImagePro Plus software package (version 6). 16. Analyze the read-through efficiency in different tissues based on the GFP39TAA expression recovery (Fig. 8). 3.9 Generation and Characterization of trans/mdx Mice

1. Mate healthy 4-week-old male transgenic mice with female mdx mice in a 1:2 ratio. When the female mice are pregnant, place them in a separate cage to breed the offspring, respectively (see Note 9).

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Fig. 8 Expression of GFP39TAA fluorescent proteins in different tissues of transgenic mice in a strictly NAEKdependent manner

2. According to Mendel’s law of heredity, all male offspring exhibit symptoms since DMD is a rare human X-chromosomelinked muscular disease. Therefore, only the PylRS–tRNA transgene needs to be further identified. 3. Label 3-week-old offspring male mice and clip their tails, extract mouse tail DNA, and set up PCR reactions according to Subheading 3.4. The positive offspring are collected based on the results of agarose gel electrophoresis. 4. Divide 4-week-old positive trans/mdx mice into two groups, with one group of mice being given a 1-week intraperitoneal injection of NAEK and the other group of mice being given a 1-week intraperitoneal injection of saline. Four-week-old normal mice, transgenic mice, and mdx mice are also grouped as controls. 5. Collect the muscle tissues of each group of mice, respectively, and the expression of PylRS and GFP is detected by western blot according to Subheading 3.5. The expression of PylRS is measured indirectly via its fusion myc-tag (Fig. 9a, b). 3.10 Restoration of Dystrophin in trans/ mdx Mice

1. Isolation of tibialis anterior muscles in positive trans/mdx mice after 0, 1, 2, 4, 6, and 8 weeks of NAEK intraperitoneal administration (see Note 10). 2. Fix tissue pieces from different mice in 4% PFA at room temperature for 1 h. 3. Wash fixed tissues with PBS three times. 4. Immerse tissues in 30% (w/v) sucrose overnight. 5. Embed tissues in optimal cutting temperature (OCT) compound (Tissue-Tek) and freeze them at -80 °C. 6. Cryo-sectioning of embedded tibialis anterior muscle tissues at -20 °C using a cryostat to obtain serial 12 μm sections. 7. Block cryosections with the blocking buffer for immunostaining for 30 min.

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Fig. 9 PylRS expression (a) and NAEK-dependent expression of GFP39TAA fluorescent proteins (b) and restoration of dystrophin (c) in tibialis anterior muscles of trans/mdx mice

8. Incubate the sections with anti-dystrophin antibody diluted in blocking buffer overnight at 4 °C. 9. Incubate the sections with secondary goat anti-rabbit IgG Alexa Fluor 594 at room temperature for 1 h. 10. Stain the sections with 0.5 μg/mL Hoechst and mount them in mounting medium (e.g., PBS). 11. Photograph stained sections under a Nikon Ti-S microscope and analyze the expression and distribution of dystrophin (Fig. 9c).

4

Notes 1. The concentration of NAEK solution can be adjusted if you need. Ensure that the pH of the solution is about 7.0 and the

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injection volume is no more than 750 μL. The pH of injected solutions would influence the cell viability significantly, especially the low pH. Please adjust the pH of solutions to about 7.0 before use. 2. The design of expression vectors is important for efficient PylRS–tRNA pair expression in vitro and in vivo. On the other hand, the inefficiency is mainly due to inappropriate promoters for aaRS and tRNA, too few copies of tRNA, or inefficient cassette construction. Strong Pol II gene promoter (CAG, EF1a, etc.) and Pol III gene promoter (7sk, U6, etc.) are recommended to be used, while you should exclude the possibility of “promoter shutdown” occurring in your interested cells or tissues at first. Using more copies of tRNA could enhance the read-through efficiency; however, a tRNA array that needs to be cleaved by RNase Z and RNase P would reduce the expression [17, 18]; we recommend four copies of tRNA to be driven by four Pol III gene promoters, respectively, instead of four copies of tRNA to be driven by one Pol III gene promoter. 3. Make sure that the linearized DNA is of high purity; for absorbance, A260/A280 should be 1.8–2.0, and A260/A230 should be 2.2–2.6. The low quality of linearized DNA would reduce the efficiency of gene targeting and even influence embryo development. 4. The mouse tail should be fully dissolved before DNA extraction. Scale the lysis reaction system if the tail is too long. The lysate mixture would be in orange to brown color, while it appears black due to mouse hairs stuck to the tail. 5. The quality of mouse tail DNA is mainly depending on the DNA adsorption columns used in extraction. We recommend using a specific tissue genome DNA extraction kit. 6. In this method, we use a prepared PCR mixture to set up reactions easily. You can adjust the amounts of each reagent, dNTPs, Mg2+, etc., in the PCR system in accordance with the DNA polymerase you are using. We recommend using less than 1.5 μL of forward/reverse primer (10 μM) in a 20 μL system, for excessive primers would result in unspecific amplification. 7. For NAEK used in this method, 50 mg every 2 days is the optimal dose for efficient protein restoration and without significant side effect. If you want to use other ncAAs, set a dose gradient of 10, 20, 30, 40, and 50 mg for injection, record, and analyze the survival of the transgenic mice after injection, to determine the optimal dose. 8. The electroblotting for large proteins is more difficult and inappropriate conditions would result in loss and even disappearance of the specific band. Lower voltage with longer

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electroblotting time could obtain a clearer protein band. We recommend conducting a preexperiment to find an optimal voltage in your laboratory. 9. Since the female mdx mice usually exhibit progressive muscle weakness and shortened life spans, you can mate one transgenic male with three or four mdx females. In addition, mdx female mice are likely to die during pregnancy or delivery; do take care to keep them warm and provide nutritious food. 10. To evaluate the therapeutic efficacy for DMD in mice, we measure the dystrophin restoration at six time points (0, 1, 2, 4, 6, 8 weeks). For investigating other nonsense mutation diseases, you could choose other time points for collecting tissues according to the progress of disease.

Acknowledgements This work was supported by the National Science and Technology Major Projects for Major New Drugs Innovation and Development (2018ZX09711003-001-003 and 2018ZX09J18114) of China and State Key Laboratory of Natural and Biomimetic Drugs to Q.X. References 1. Young DD, Schultz PG (2018) Playing with the molecules of life. ACS Chem Biol 13:854– 870 2. de la Torre D, Chin JW (2021) Reprogramming the genetic code. Nat Rev Genet 22:169– 184 3. Wan W, Tharp JM, Liu WR (2014) PyrrolysyltRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim Biophys Acta (BBA) Proteins Proteomics 1844:1059–1070 4. Shi N, Tong L, Lin H et al (2022) Optimizing eRF1 to enable the genetic encoding of three distinct noncanonical amino acids in mammalian cells. Adv Biol 6(11):2200092 5. Ernst RJ, Krogager TP, Maywood ES et al (2016) Genetic code expansion in the mouse brain. Nat Chem Biol 12:776–778 6. Krogager TP, Ernst RJ, Elliott TS et al (2018) Labeling and identifying cell-specific proteomes in the mouse brain. Nat Biotechnol 36:156–159 7. Bladen CL, Salgado D, Monges S et al (2015) The TREAT-NMD DMD global database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat 36:395– 402

8. Fujikura K (2016) Premature termination codons in modern human genomes. Sci Rep 6:22468 9. Godfrey C, Muses S, McClorey G et al (2015) How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse. Hum Mol Genet 24:4225–4237 10. Karijolich J, Yu Y-T (2014) Therapeutic suppression of premature termination codons: mechanisms and clinical considerations (review). Int J Mol Med 34:355–362 11. Shi N, Yang Q, Zhang H et al (2021) Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids. Nat Biomed Eng 6:1–12 12. Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308 13. Landel CP (1991) The production of transgenic mice by embryo microinjection. Genet Anal Tech Appl 8:83–94 14. Murphy D (1993) Vasectomizing a mouse. Methods Mol Biol 18:137–140 15. Murphy D (1993) Mating mice. Methods Mol Biol 18:131–134

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16. Chu VT, Weber T, Graf R et al (2016) Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol 16:4 17. Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability

with the endogenous tRNA-processing system. Proc Natl Acad Sci 112:3570–3575 18. Phizicky EM, Hopper AK (2010) tRNA biology charges to the front. Genes Dev 24:1832– 1860

INDEX A

G

Antibody .................................. 21, 22, 25–28, 30, 32–38, 42, 64, 118, 122, 123, 125, 126, 128, 194–196, 205, 208, 209, 220, 221, 227, 267, 279, 281 Artificial photoenzyme..............................................42, 44

Gene randomization ...................................................9, 11 Genome editing ............................................................ 170 G protein-coupled receptor (GPCR).................. 201, 202 Green fluorescent protein (GFP) .....................42, 56, 71, 73, 79, 102, 172, 174, 175, 182, 195, 216, 220, 227, 234, 238, 239, 280

B Bioluminescence resonance energy transfer (BRET) ..................................................... 201–211 Bioorthogonal conjugation ......................... 5, 22, 55, 88, 118, 126, 191, 216 1,2-aminothiol ............................................... 191–198 copper-catalyzed azide-alkyne cycloaddition (CuAAC) ................................. 109–110, 113, 126 inverse electron-demand Diels–Alder cycloaddition ................................... 118, 126, 202 oxime ligation................................................... 95, 110 strain-promoted azide-alkyne cycloaddition (SPAAC) .............................................................. 94 tetrazine ligation ...........................216, 221, 222, 231

C Chemogenetics.............................................................. 216 Confocal microscopy ........................................... 194, 216 CRISPR-Cas9....................................................... 170–172

D Danio rerio, see Zebrafish Deubiquitinase ..........................................................55–65 Directed evolution ............................................... 182, 183

E Embryo .............................. 247–261, 268, 274–276, 282 Enzyme-linked immunosorbent assay (ELISA)........... 33, 119, 122, 125, 128, 205, 209 Escherichia coli transformation ....................................... 80

F Flow cytometry ............................................ 33, 164, 182, 186, 187, 189, 235, 239 Fo¨rster resonance energy transfer (FRET)..................................................55, 56, 202

H Histone ..................... 131–133, 140, 142, 143, 148, 154

I Intein .........................................................................69–85 In vitro transcription................................... 250, 253, 260

K Kinase..........................................215, 216, 219, 222, 230 Knock-in ..................................... 171, 173–179, 268, 270

L Lentivirus ....................................................................... 164 Luciferase...................................... 73, 202, 249, 256, 261

M Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)............ 4, 28, 102–104, 111, 118, 234 Microinjection in mouse ................................................ 268, 273, 275 in zebrafish .............................................252, 254–255 Molecular dynamics ...................................................... 203 Mouse ......................................................... 132, 159, 170, 173, 192–196, 205, 209, 221, 265–283

N Nonsense mutation disease Duchenne muscular dystrophy (DMD) ............... 266, 268, 278, 280

P Phage display ............................................... 117, 118, 127 Photocaged tyrosine (pcY) ...... 22, 24, 26–28, 30–33, 38

Yu-Hsuan Tsai and Simon J. Elsa¨sser (eds.), Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2676, https://doi.org/10.1007/978-1-0716-3251-2, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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286 Index

Photosensitizer ..........................................................41–51 PiggyBac transposase .................................................... 179 Post-translation modifications (PTMs)...................................................... 102, 131 glutamine methylation ............................. 87, 147–155 lysine acetylation ..................................................... 131 lysine benzoylation......................................... 131–144 tyrosine sulfation ............................................ 233, 234 Protease ........................................................ 56, 102, 198, 220, 226, 249, 250, 258, 275, 276 Pyrrolysyl-tRNA synthetase Desulfitobacterium hafniense....................................... 4 Methanomethylophilus alvus .................. 102, 103, 105 Methanosarcina barkeri....................................4–7, 17, 154, 160, 248 Methanosarcina mazei................................. 4, 5, 7, 56, 63, 102, 103, 105, 160, 192, 193

Q Quadruplet codon........................................ 22, 118, 127, 160, 181, 182, 184, 186

R Recombinant protein expression in E. coli .................. 148

S Selenocysteine (Sec) ............................................... 69, 117 Sirtuin ................................ 132, 133, 138, 141, 142, 144 Stable cell line......................................164, 170, 177–179 Staudinger reduction .................................................... 248

Z Zebrafish .......................................................132, 247–261