Heterologous Expression of Membrane Proteins: Methods and Protocols [1 ed.] 1607613433, 9781607613435, 9781607613442

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METHODS

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MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

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Heterologous Expression of Membrane Proteins Methods and Protocols

Edited by

Isabelle Mus-Veteau Institut of Developmental Biology and Cancer, UMR CNRS 6543 Université de Nice-Sophia Antipolis, Parc Valrose, Nice, France

Editor Isabelle Mus-Veteau, Ph.D. Institut of Developmental Biology and Cancer, UMR CNRS 6543 Université de Nice-Sophia Antipolis Parc Valrose, Nice France [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-343-5 e-ISBN 978-1-60761-344-2 DOI 10.1007/978-1-60761-344-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2009932113 © Humana Press, a part of Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: The background art is derived from Figure 5 in Chapter 12. Printed on acid-free paper Humana Press is a part of Springer Science+Business Media (www.springer.com)

Preface Membrane proteins account for roughly 30% of all open reading frames in fully sequenced genomes. These proteins are of central importance to living cells. They are required for transport processes, sensing changes in the cellular environment, transmission of signals, and control of cell–cell contacts. These proteins are implicated in numerous pathologies, like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer disease, but studies of these and other disorders are hampered by a lack of information about the proteins involved. Knowing the structure of membrane proteins is an essential prerequisite for understanding how these proteins function and, further, how their functions can be modified by small molecules. This is of paramount importance in the pharmaceutical industry, which produces many drugs that bind to membrane proteins (50% of all drug targets are G protein-coupled receptors [GPCRs]), and recognizes the potential of many recently identified GPCRs, ion channels and transporters as targets for future drugs. However, whereas high-resolution structures are available for myriad soluble proteins (more than 42,000 in the Protein Data Bank), three-dimensional (3D) structures have so far been obtained for only 204 membrane proteins, the majority of which are from prokaryotic organisms, with only 25 from mammalian membrane proteins (see http:// blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). The first membrane proteins were crystallized owing to their natural abundance, circumventing all the difficulties associated with overexpression. However, the majority of medically and pharmaceutically relevant membrane proteins are present in tissues at very low concentration, making overexpression of recombinant membrane proteins in heterologous cells suitable for largescale production a prerequisite for structural studies. The development of heterologous expression systems capable of delivering high-quality recombinant protein for structural studies remains an essential goal. In 2005, the two first atomic structures of recombinant mammalian membrane proteins expressed in yeast were resolved. Since that time, extensive optimization of heterologous expression systems has begun to bear fruit, and 11 eukaryotic membrane protein structures have been solved to high resolution using recombinant material. This volume proposes an overview of different heterologous expression systems that produce an adequate amount of membrane proteins for structural analysis. Methods and protocols are described for each heterologous expression system proposed. Some chapters of this volume treat membrane protein solubilization, purification and instability in solution and comment on the strategies that allowed the determination of the structure of the first heterologously expressed mammalian membrane proteins. Isabelle Mus-Veteau Nice, France

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Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 3

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Heterologous Expression of Membrane Proteins for Structural Analysis . . . . . . . . Isabelle Mus-Veteau Production of Membrane Proteins in Escherichia coli and Lactococcus lactis . . . . . . Eric R. Geertsma and Bert Poolman Mammalian Membrane Receptors Expression as Inclusion Bodies in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bernard Mouillac and Jean-Louis Banères Expression of Membrane Proteins at the Escherichia coli Membrane for Structural Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manuela Zoonens and Bruno Miroux Membrane Protein Expression in Lactococcus lactis . . . . . . . . . . . . . . . . . . . . . . . . . . Annie Frelet-Barrand, Sylvain Boutigny, Edmund R.S. Kunji, and Norbert Rolland Heterologous Expression of Human Membrane Receptors in the Yeast Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Olivier Joubert, Rony Nehmé, Michel Bidet, and Isabelle Mus-Veteau Mammalian Membrane Protein Expression in Baculovirus-Infected Insect Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Céline Trometer and Pierre Falson Expression of membrane proteins in Drosophila melanogaster S2 Cells: Production and Analysis of a EGFP-Fused G Protein-Coupled Receptor as a Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karl Brillet, Carlos A. Pereira, and Renaud Wagner Membrane Protein Expression in the Eyes of Transgenic Flies. . . . . . . . . . . . . . . . Valérie Panneels and Irmgard Sinning Expression of Mammalian Membrane Proteins in Mammalian Cells Using Semliki Forest Virus Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kenneth Lundstrom Membrane Protein Expression in Cell-Free Systems . . . . . . . . . . . . . . . . . . . . . . . Birgit Schneider, Friederike Junge, Vladimir A. Shirokov, Florian Durst, Daniel Schwarz, Volker Dötsch, and Frank Bernhard Practical Considerations of Membrane Protein Instability during Purification and Crystallisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Christopher G. Tate Membrane Protein Solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Katia Duquesne and James N. Sturgis

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Amphipols and Fluorinated Surfactants: Two Alternatives to Detergents for Studying Membrane Proteins In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Cécile Breyton, Bernard Pucci, and Jean-Luc Popot 15 Heterologous Expression and Affinity Purification of Eukaryotic Membrane Proteins in View of Functional and Structural Studies: The Example of the Sarcoplasmic Reticulum Ca2+-ATPase. . . . . . . . . . . . . . . . . . . 247 Delphine Cardi, Cédric Montigny, Bertrand Arnou, Marie Jidenko, Estelle Marchal, Marc Le Maire, and Christine Jaxel Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

Contributors BERTRAND ARNOU • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France JEAN-LOUIS BANÈRES • Institut des Biomolécules Max Mousseron, Université Montpellier I, Université Montpellier II, Faculté de Pharmacie, Montpellier, France FRANK BERNHARD • Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany MICHEL BIDET • Institut of Developmental Biology and Cancer, UMR CNRS 6543, Université de Nice-Sophia Antipolis, Parc Valrose, Nice, France SYLVAIN BOUTIGNY • Laboratoire de Physiologie Cellulaire Végétale, Grenoble, France; and CEA, DSV, iRTSV, Grenoble, France; INRA, Grenoble, France; Université Joseph Fourier, Grenoble, France CÉCILE BREYTON • Institut de Biologie Structurale, UMR 5075 CNRS/CEA/UJF, 41, rue Jules Horowitz, 38027 Grenoble France KARL BRILLET • Dpt Récepteurs et des Protéines Membranaires, Illkirch, France DELPHINE CARDI • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France VOLKER DÖTSCH • Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany KATIA DUQUESNE • Laboratoire d’Ingenierie des Systèmes Maromoléculaire, Institut de Biologie Structurale et Microbiologie, CNRS and Aix Marseille Université, Marseille, France FLORIAN DURST • Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany PIERRE FALSON • Laboratoire des Protéines de Résistance aux Agents Chimiothérapeutiques (LPRAC), Institut de Biologie et Chimie des Protéines (IBCP), Unité Mixte du Centre National de la Recherche Scientifique (CNRS) et de l’Université Lyon I, Institut Fédératif de Recherches Lyon, France ANNIE FRELLET-BARRAND • Institut de Biologie Structurale, UMR 5075 CNRS/CEA/ UJF, 41, rue Jules Horowitz, 38027 Grenoble France ERIC R. GEERTSMA • Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Netherlands Proteomics Centre (NPC), and Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands CHRISTINE JAXEL • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France MARIE JIDENKO • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France OLIVIER JOUBERT • Université Henri Poincaré- Nancy 1, Faculté de Pharmacie, 5 rue Albert Lebrun, BP 80403, F-54001 NANCY CEDEX FRIEDERIKE JUNGE • Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany ix

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EDMUND R.S. KUNJI • The Medical Research Council, Dunn Human Nutrition Unit, Cambridge, UK MARC LE MAIRE • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France KENNETH LUNDSTROM • PanTherapeutics, Lutry, Switzerland ESTELLE MARCHAL • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France BRUNO MIROUX • Université Paris-7, Laboratoire de Biologie Physico-Chimique des Proteines Membranaires, Institut de Biologie Physico-Chimique, Paris, France CÉDRIC MONTIGNY • CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France BERNARD MOUILLAC • Institut de Génomique Fonctionnelle, Montpellier, France; INSERM U661, Montpellier, France; Université Montpellier I, Montpellier, France; Université Montpellier II, Montpellier, France ISABELLE MUS-VETEAU • Institut of Developmental Biology and Cancer, UMR CNRS 6543, Université de Nice-Sophia Antipolis, Parc Valrose, Nice, France RONY NEHMÉ • Institut of Developmental Biology and Cancer, UMR CNRS 6543, Université de Nice-Sophia Antipolis, Parc Valrose, Nice, France CARLOS A. PEREIRA • Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, Brasil VALÉRIE PANNEELS • Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany BERT POOLMAN • Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Netherlands Proteomics Centre (NPC), and Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands JEAN-LUC POPOT • Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, CNRS and Université Paris-7, Institut de Biologie Physico-Chimique, Paris, France BERNARD PUCCI • Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels, Université d’Avignon et des Pays de Vaucluse, Faculté des Sciences, Avignon, France DANIEL SCHWARZ • Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany BIRGIT SCHNEIDER • Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany VLADIMIR A. SHIROKOV • Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia IRMGARD SINNING • Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany JAMES N. STURGIS • Laboratoire d’Ingenierie des Systèmes Maromoléculaire, Institut de Biologie Structurale et Microbiologie, CNRS and Aix Marseille Université, Marseille, France CHRISTOPHER G. TATE • MRC Laboratory of Molecular Biology, Cambridge, UK CÉLINE TROMETER • Laboratoire des Protéines de Résistance aux Agents Chimiothérapeutiques (LPRAC), Institut de Biologie et Chimie des Protéies

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(IBCP), Unité Mixte du Centre National de la Recherche Scientifique (CNRS) et de l’Université Lyon, Institut Fédératif de Recherches, Lyon, France NORBERT ROLLAND • Laboratoire de Physiologie Cellulaire Végétale, CNRS, Grenoble, France; and CEA, DSV, iRTSVGrenoble, France; INRA, Grenoble, France; Université Joseph Fourier, Grenoble, France RENAUD WAGNER • Dpt Récepteurs et des Protéines Membranaires, Illkirc, France MANUELA ZOONENS • Université Paris-7, Laboratoire de Biologie Physico-Chimique des Proteines Membranaires, Institut de Biologie Physico-Chimique, Paris, France

Chapter 1 Heterologous Expression of Membrane Proteins for Structural Analysis Isabelle Mus-Veteau Abstract Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are involved in numerous diseases, like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer disease. However, studies of these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, which makes heterologous expression in large-scale productionadapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the heterologous expression systems now available to produce large amounts of MPs for structural proteomics, and describes the strategies that allowed the determination of the structure of the first heterologously expressed mammalian MPs. Key words: Integral membrane proteins, heterologous expression systems, solubilization, stabilization, crystallization, 3D structure

1. Introduction Integral membrane proteins (MPs) account for roughly 30% of all open reading frames in fully sequenced genomes (1). These proteins are of central importance to living cells. They are required for transport processes, sensing changes in the cellular environment, transmission of signals, and control of cell–cell contacts. MPs are implicated in numerous pathologies like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer disease, but studies of these and other disorders are hampered by a lack of information about the proteins involved. Knowing the structure of MPs is an essential prerequisite for understanding how MPs function and, further, how their functions can be modified by small molecules. This is of paramount

I. Mus-Veteau (ed.), Heterologous Expression of Membrane Proteins, Methods in Molecular Biology, vol. 601 DOI 10.1007/978-1-60761-344-2_1, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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importance in the pharmaceutical industry, which produces many drugs that bind to MPs (50% of all drug targets are G proteincoupled receptors (GPCRs) (2)), and recognizes the potential of many recently identified GPCRs, ion channels and transporters as targets for future drugs. However, whereas high-resolution structures are available for myriad soluble proteins (>42,000 in the NCBI database Protein Data Bank), three-dimensional (3D) structures have so far been obtained for only 204 integral MPs, the majority of which are from prokaryotic organisms, with only 25 from mammalian MPs (see http://blanco.biomol.uci.edu/ Membrane_Proteins_xtal.html). The first MPs were crystallized thanks to their natural abundance, circumventing all the difficulties associated with overexpression (3–6)). The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, making overexpression of recombinant MPs in heterologous cells suitable for large-scale production a prerequisite for structural studies. The development of heterologous expression systems capable of delivering high-quality recombinant protein for structural studies remains an essential goal. In 2005, the two first atomic structures of recombinant mammalian MPs expressed in yeast were resolved (7, 8)). Extensive optimization of heterologous expression systems (see (9) for review) has begun to bear fruit, and 11 eukaryotic MPs structures have been solved to high resolution using recombinant material (10–21). This review introduces the different heterologous expression systems available to produce amounts of MPs adequate for structural analysis, for which detailed methodologies are described in the following chapters of this volume, and comments on the strategies that allowed the determination of the structure of the first heterologously expressed eukaryotic MPs.

2. Heterologous Expression Systems 2.1. Bacteria 2.1.1. Prokaryotic Membrane Protein Expression in Bacteria

Due to the development of efficient genetic tools, simplicity and low cultivation cost, Escherichia coli is considered the most popular host for the overproduction of MPs. However, E. coli often has difficulties with the expression, folding, stability and assembly of eukaryotic MPs. A convenient strategy to obtain structural and functional information on a certain MP that is not abundant in any natural source involves the screening of prokaryotic homologues, which can often be expressed in bacteria in large quantities. To rapidly establish an over-expression system, several homologues, types and location of affinity tags necessary for purification and expression strains need to be screened. For this, high-throughput procedures have been developed. The large number of plasmids required for this strategy implicates the development of efficient

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cloning procedures such as ligation-independent cloning (LIC) (22). Following initial screening for expression, systematic optimization of the production conditions to increase the amount of well-folded protein inserted into the membrane is often required. This process, which involves time-consuming steps such as the isolation of membrane vesicles and activity assays, has been greatly accelerated by the use of green fluorescent protein (GFP) fused to the C-terminal end of the target protein. Since proper maturation of GFP in E. coli depends on the correct folding of its fusion partner, only correct folding of the target protein leads to a fluorescent fusion protein (23). Geertsma and Poolman detail in Chapter 2 of this volume methodology to rapidly establish and optimize prokaryotic MP expression in E. coli and Lactococcus lactis. This method cannot easily be extended to mammalian MPs because these proteins are mostly expressed in inclusion bodies (IBs) in E. coli, from which they are usually impossible to purify under non-denaturing conditions. Therefore, other bacteria like L. lactis, or derivatives of E. coli BL21 (DE3) selected to grow to high-saturation cell density and to overproduce proteins without toxic effect for the host cell, have been successfully used for production of some eukaryotic MPs. 2.1.2. Production of Eucaryotic Membrane Proteins in E. coli Inclusion Bodies

One phenomenon frequently observed in E. coli is that many heterologous proteins become incorrectly folded and accumulate in the cytoplasm as insoluble aggregates called inclusion bodies (IBs). As described by Margreiter and co-workers (24), IBs are observed as refractive particles either in the cytoplasm or in the periplasmic space if the heterologous protein is engineered for secretion. They can grow larger than 1 mm in diameter, a large proportion of a single bacterial cell, and thus are clearly visible under a microscope. IBs contain mostly recombinant protein (up to 99%) but also chaperones and membrane fragments. Because IBs are usually mechanically stable, they can be isolated from cells and separated by centrifugation. In many cases, IB formation strongly boosts MP production up to several thousand-fold without toxicity for bacteria, which is particularly interesting for structural approaches. MPs accumulated in IBs are protected against degradation by proteolysis. However, to recover the entrapped recombinant proteins, IBs must be dissolved by highly concentrated chaotropic agents like 8M urea or 6M guanidine hydrochloride. The bottleneck of this strategy is to establish efficient refolding protocols after purification to obtain pure functional MPs. Several MPs have been successfully refolded after purification from IBs: the light-harvesting complex (LHC) (25), diacylglycerol kinase (DAGK) (26), and the leukotriene B4 receptor BLT1 (27, 28). In Chapter 3 of this volume, Mouillac and Banères describe general trends for expression and purification of membrane receptors from E. coli IBs, as well as a general screening assay for refolding conditions.

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2.1.3. E. coli Strains for Functional Expression of Eukaryotic Membrane Proteins

Aggregation of foreign MPs in IBs can be reduced and targeting to the membrane favoured by using low-copy-number plasmids with weak promoters and fusion proteins between the MP of interest and a partner known to target the membrane, optimizing the cell growth conditions and selecting host mutants resistant to toxicity. MP expression at the E. coli membrane can be increased in specific mutant strains. C41 and C43 (DE3) are mutant hosts that allow synthesis of some MPs at high levels (29). Expression of certain MPs in the C43 (DE3) strain appears to be accompanied by proliferation of intracellular membrane, providing more space for MP insertion (30). Proteolysis of MPs can be decreased using BL21 derivative strains with low proteolytic activity. Zoonens and Miroux describe in Chapter 4 of this volume a protocol for mammalian MP expression at the E. coli membrane.

2.1.4. Eukaryotic Membrane Protein Expression in L. lactis

Lactococcus lactis is a gram-positive lactic bacterium traditionally used for the production of lactic acid in fermented food products. Genetic engineering tools and molecular characterization have been developed on Lactococcus (31–33), and now this bacterium is widely used in biotechnology for large-scale overproduction of heterologously expressed proteins. As an expression host, L. lactis presents advantages over E. coli: It is a food-grade organism, does not produce endotoxins, possesses low protease activity, does not form IBs, and has only one membrane. In 2005, Kunji and coworkers reported the overproduction of 11 mitochondrial transport proteins from yeast in a functional state in the L. lactis cytoplasmic membrane (34). A. Frelet-Barrand and collaborators report in Chapter 5 of this volume detailed protocol for the expression of MPs in L. lactis using the nisin-inducible controlled gene expression (NICE) system, which allows fine control of gene expression based on the auto-regulation mechanism of the bacteriocin nisin. Despite the advantages offered by bacteria, success rates of eukaryotic MP expression for structural proteomics in E. coli are between 10% and 30% compared with 40–70% in eukaryotic systems (2, 35).

2.2. Yeast

This simple eukaryotic cell is a multi-purpose host, performing many of the post-translational modifications seen in higher eukaryotic cells (glycosylation, disulphide bond formation and proteolytic processing), combined with the ease of growing a large volume of cells in a short period of time. Cloning, functional expression, identification of interacting partners, mutagenesis of the protein or its partners and overexpression for biophysical analysis can all be achieved in yeast (36). However, yeast glycosylation patterns and lipid composition of membranes (no endogenous cholesterol) do differ from those of mammalian cells, and this can have important consequences on activity, folding, trafficking,

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stability and structural studies of MPs (8, 19, 37, 38). Exciting possibilities also exist for the exploitation of yeast genetics to tailor yeast strains so that they are optimized for the expression of mammalian MPs, like yeast with humanized glycosylation machineries that have become available recently (39). Two yeast species have so far been used successfully for heterologous expression of mammalian MPs: Saccharomyces cerevisiae and Pichia pastoris. From the 11 atomic structures of mammalian MPs derived from a heterologous overproduction source, 5 are derived from yeast: 1 from S. cerevisiae, the rabbit sarcoplasmic reticulum Ca2+-ATPase (SERCA1A) (7); 3 from P. pastoris, the rat potassium channel (8), the chimera potassium channel Kv1.2/Kv2.1 (15) and the human LTC4 synthase (16). The structure of the last protein was also obtained from its expression in S. Pombe (10). 2.2.1. Membrane Protein Expression in the Yeast S. cerevisiae

Saccharomyces cerevisiae has been successfully used to functionally express and purify several recombinant mammalian MPs, like the human P-glycoprotein (40), the rat vesicular monoamine transporter (41), the human receptor of the Hedgehog pathway (42), the Aquaporin AQP0 (43) and the rabbit SERCA1a Ca2+-ATPase (adenosine triphosphatase) that gave the first atomic structure of a heterologously expressed mammalian MP (7). A lot of work has been done on S. cerevisiae to understand the parameters involved in MP expression, and many strains are available. The expression vectors used are 2 m multicopy plasmids with the inducible GAL1 promoter or the strong constitutive promoter from the yeast plasma membrane ATPase (PMA1). Depending on the conditions, the human P-glycoprotein expression reaches 8% of total MPs using a proteasedeficient strain of S. cerevisiae, with a level of expression comparable to that of mammalian cells (COS cells and Human Embryonic Kidney 293 cells) at only a fraction of the cost, time and effort (40). It has been shown for several MPs that the functional expression in S. cerevisiae can be enhanced by modification of certain codons in the translation initiation region to codons preferred by S. cerevisiae and by reducing the temperature to 16°C (42, 44, 45). In Chapter 6 of this volume, Joubert and co-workers describe a protocol to functionally express human MPs in the yeast S. cerevisiae.

2.2.2. Membrane Protein Expression in the Yeast Pichia pastoris

The methylotrophic yeast P. pastoris has been used successfully as a tool for large-scale recombinant soluble protein production (46). Pichia pastoris is a poor fermenter, a major advantage relative to S. cerevisiae. In cultures with high cell density, ethanol (a product of S. cerevisiae fermentation) rapidly builds to toxic levels, limiting further growth and foreign protein production. With its preference for respiratory growth, P. pastoris can be cultured at extremely high densities (500 OD600 U/mL) in the controlled environment of a fermenter. Also, P. pastoris is less likely to hyperglycosylate recombinant MPs than S. cerevisiae and foreign genes are stably integrated

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in single or multiple copies behind the AOX1 (alcohol oxidase 1) promoter, one of the strongest and most tighly regulated promoters known. Pichia pastoris has been used to produce many eukaryotic MPs in high yield (47), including the rat potassium channel and the human LTC4 synthase used for atomic structure determination (8, 16), and even several GPCRs (42, 48). 2.3. Insect Cells

Although more expensive to culture than yeast, insect cells remain simpler to maintain in large-scale culture than mammalian cells. Furthermore, even if insect cells glycosylate poorly and essentially with mannose (49) and their membrane presents a low level of cholesterol, they offer a protein-processing machinery and membrane composition closer to those of mammalian cells than yeast (50).

2.3.1. Mammalian Membrane Protein Expression in Baculovirus Infected Insect Cells

The baculovirus expression vector system (BEVS) is based on replacement of a late, non-essential, viral gene coding for the polyhedron with a gene of interest. BEVS allows cloning and expression of recombinant proteins in insect cells of Lepidopterans such as Spodoptera frugiperda (Sf9, Sf21) and Trichoplusia ni (Hi5). Recombinant baculovirus and insect cells have been essayed for expression of several GPCRs (51), and were used to produce the β2- and the β1-adrenergic receptors employed to determine the X-ray structures (11, 18, 21). This system was also used to produce the aquaporin AQP4 protein employed in high-resolution electron crystallographic studies (13) and the acid-sensing ion channel 1 that gave the X-ray atomic structure (14). Trometer and Falson detail in Chapter 7 of this volume the experimental procedure they developed to produce a human MP, the breast cancer resistance protein (BCRP), by using the Bac-to-Bac baculovirus-based expression system.

2.3.2. Mammalian Membrane Protein Expression in Transiently or Stably Transfected Drosophila Schneider 2 Cells

The Drosophila Schneider 2 (S2) cells are immortalized nontumorigenic cells isolated from primary cultures of Drosophila melanogaster embryos (52). One specific characteristic of S2 cells is their ability to grow to high cell densities, as high as 3 × 107 cells/mL, which is tenfold higher than what can be attained with other insect cells, like Sf9 (53). Moreover, S2 cells can be transiently or stably transfected using a non-lytic plasmid-based system that provides additional benefits over virus-infected insect systems. S2 cells possess all the mammalian-like cell machineries for gene expression, protein processing and trafficking, including posttranslational modifications that may be critical for proper maturation, localization and function of target protein (54). Although not yet fully explored and poorly used, S2 cells have been shown to be suitable hosts for the expression of large amounts of several recombinant mammalian MPs like GPCRs (55–59). In Chapter 8 of this volume, Brillet and co-workers present a series of protocols allowing handling S2 cell expression system through the example of a human GPCR fused to the enhanced GFP.

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2.4. Drosophila Eyes

The Sinning lab developed the expression of recombinant MPs in the eyes of transgenic Drosophila melanogaster as a new, alternative method for overexpression. This idea was based on the observation that the rhodopsin, the first GPCR from which an X-ray structure was obtained in 2000 (3), is densely packed in retina membranes, where it is targeted to the specialized membrane stacks in the photoreceptor cells, termed rhabdomeres in Drosophila (60). Using developmental drivers (promoters) that activate the expression of specific genes at certain stages of development, it is possible to express MPs that localise to these specialized membranes. Expression in photoreceptor cells provides a number of advantages compared to conventional expression systems. In these membranes, MPs are protected from proteases. Internal membranes are reduced to a minimum, and about 90% of the membranes are plasma membrane. This avoids or minimizes the problem of proteolysis and of mistargeted or incompletely matured protein. Several GPCRs and transporters not only from Drosophila but also from mammals were successfully expressed in the fly eyes demonstrating the great potential of this method (61). In Chapter 9 of this volume, Panneels and Sinning present a protocol of expression of MPs in Drosophila eyes using GFP fusion constructs that allow monitoring expression, purification and localization of the proteins.

2.5. Mammalian Cells

Mammalian cells clearly offer the most native cellular environment for the expression of mammalian MPs. There are some mammalian MPs for which heterologous expression of functional protein is limited to mammalian cells in culture, as is the case with many GPCRs (62). For at least some recombinant mammalian MPs, mammalian cells provide the greatest amount of functional protein (2, 63, 64). But, generation of milligram quantities required for crystallization trials with these cells is expensive and labour-intensive and post-translational modifications like glycosylation can be highly heterogeneous. However, the first structure of a recombinantly produced GPCR was obtained from a rhodopsin mutant expressed in mammalian cells (19). Mammalian expression systems have relied on transiently transfected cells, the isolation of stable transformants or the use of viral vectors.

2.5.1. Production of Membrane Proteins in Stable or Transient Transformant Mammalian Cells

Efforts have focused on careful optimization of culture conditions and media components with respect to transfection efficiency and protein expression levels. As a result, together with the development of the serum-free media and of relatively cheap transfection reagents, large-scale transient transfection (LSTT) of mammalian cells has achieved growing acceptance as an alternative expression method (65). The transient expression of the recombinant rhodopsin in COS-1 cells allowed production of enough stable protein to determine the first atomic structure of a recombinantly produced GPCR (19).

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2.5.2. Production of Membrane Proteins in Mammalian Cells Infected by the Semliki Forest Virus

Among the most widely viral vectors used for MP expression in mammalian cells is the Semliki Forest virus (SFV). The broad host range of SFV allows transduction of a large number of mammalian cell lines and primary cell cultures. Expression of GPCRs and ion channels from SFV vectors has provided pharmacologically functional MPs, has demonstrated coupling of G proteins to GPCRs (66) and has shown electrophysiological responses to the ion channel (67). Efficient infection of both adherent and suspension cultures of mammalian cells has allowed scale-up of recombinant protein production in spinner and roller flasks as well as in bioreactors, which has allowed the production of hundreds of milligrams of recombinant MPs (2, 68, 69). In Chapter 10 of this volume, Lundstrom describes a protocol for expression of recombinant mammalian MPs in mammalian host cells applying SFV vectors.

2.6. Cell-Free System

The cell-free expression system mimics the natural cytoplasmic cell environment yet it remains independent from the requirements and sensitivity of living cells. The tolerance of the cell-free expression system for a wide variety of supplements offers a wealth of perspectives for the high-level production of proteins that would be difficult or even impossible to obtain in cellular hosts, like some mammalian integral MPs. In principle, almost any compounds that might be beneficial for the stabilization or the folding of a recombinant protein like protease inhibitors, cofactors, substrates or any kind of ligands can be added directly into the cell-free system. Problems of toxicity, instability or protein folding can therefore be specifically addressed in many cases. In particular, the addition of detergents into the cell-free system offers the possibility to synthesize integral MPs by providing an artificial hydrophobic environment that prevents the aggregation of freshly translated MPs and supports their functional folding in the detergent micelles (70–74). This strategy was successful for several integral MPs, including GPCRs (75–78). Liposomes can also be added in the cell-free reaction allowing the synthesized MPs to directly integrate into the lipidic bilayer (79–81). In Chapter 11 of this volume, Schneider and co-workers describe the basic protocols for the general production of MPs in a cell-free expression system using E. coli extracts.

3. Toward Mammalian Membrane Protein Crystallization

Crystallization requires pure MP that is chemically and conformationally homogeneous, stable at relatively high concentrations for a long period and available in milligram amounts.

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3.1. Membrane Protein Solubilization and Stability

Purification of MPs requires the use of detergents to extract them from the membrane. MPs are then in aqueous solution in complex with detergents and lipids. Finding a suitable detergent and conditions that ensure protein homogeneity, functionality, stability and crystallization is a limiting and crucial step. Strategies have been developed to facilitate the purification of MPs in a functional form leading to new possibilities for structure determination. These strategies allowed the resolution of the three first atomic structures derived from recombinant GPCRs. Tate reviews these strategies in Chapter 12 of this volume. Scaffolds of the crystal lattice are found predominantly between the exposed, polar surfaces of proteins, while the transmembrane parts remain buried from the detergent micelle. Proteins with large extra-membranous domains are favoured, and detergents that assemble into small micelles, such as octyl-β-D-glucopyranoside (OG) or dimetyldodecylamineoxide (LDAO) are preferred in crystal trials (82). However, dodecyl-β-D-maltoside (DDM), a detergent that forms large micelles, has given rise to some structures, such as those of the acid-sensing ion channel 1 (14) or the human LTC4 synthase (16). In Chapter 13 of this volume, Duquesne and Sturgis give a general protocol for optimizing MP solubilization and indicate the major parameters that are important for reproducibility.

3.2. New Surfactant Molecules to Stabilize Membrane Proteins in Solution

A detergent efficient for the solubilization of a MP that ensures functionality and stability but is not suitable for crystallization can be exchanged during or after purification by another more suitable for crystallization. Surfactant molecules that would be more suitable than classical detergents for MP integrity, functionality and stability, like non-ionic fluorinated surfactants or amphipols, are currently under development by Pucci and co-workers (83) and Popot and co-workers (84), respectively. These molecules do not solubilize biological membranes but are able to maintain MPs that have been extracted from membrane using classical detergents in solution after exchange on an affinity column, for example. These new surfactants have been efficient for MP stability in solution (85, 86), refolding (28) and MP expression in a cell-free system (78), but as yet no MP has been crystallized in these surfactants. Breyton and co-workers describe in Chapter 14 of this volume the protocols they developed to obtain stable MPs in fluorinated surfactants and in amphipols.

3.3. Membrane Protein Purification

The purification strategy and protocol are crucial for the suitability of MPs for structural studies. The choice and the position of the affinity tags; the presence of the MP ligand, agonist or antagonist; the choice of the detergent that must be efficient for solubilization, must preserve MP integrity and must be suitable for crystallization; the possibility of detergent exchanges and the addition

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of lipids are central determinants. The purification protocol must preserve the MP integrity and homogeneity. Affinity purification allows efficient and fast purification in a few steps. This can prevent the complete removal of native lipids bound to the protein and preserve MP integrity. However, this requires expressing the target recombinant protein with one or several purification tags in its N- or C- terminus. The presence of these tags may have an effect on expression levels and on the conformational state of the protein and may affect protein crystallization (87). The incorporation of proteolytic cleavage sites that permit downstream removal of the tag can be important. The availability of a test that allows following the conformational state of the target MP during purification, such as ligand-binding measurements, is essential to establish the best purification conditions. Cardi and co-workers describe in the last chapter of this volume (Chapter 15) the protocol used to express and purify the recombinant sarcoplasmic reticulum Ca2+-ATPase from which the atomic structure was resolved.

4. The Atomic Structures of Mammalian Membrane Proteins Derived from Heterologous Expression Systems

The rabbit sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) is the first successful crystallization of mammalian MP derived from a heterologous expression system. The recombinant protein was expressed in the yeast S. cerevisiae, with a Biotin Acceptor Domain (BAD) in its C-terminus. The recombinant SERCA1a was solubilized in DDM and purified by affinity chromatography using a streptavidin-sepharose resin followed by size exclusion chromatography on which the detergent DDM was exchanged with C12E8 more suitable for crystallization. The recombinant SERCA1a crystallized in a form that is isomorphous to the native SERCA1a protein from rabbit, and the diffraction properties were similar (7). This result is important since it demonstrates that a recombinant MP produced in a heterologous expression system can adopt the same structure as the native one. At the same time, the crystal structure of a recombinant mammalian voltage-dependent potassium channel (Kv1.2) in complex with an oxidoreductase β2 regulatory subunit was solved at 2.9-Å resolution (8). Kv1.2 was mutated to eliminate the glycosylation site in the S1–S2 linker. Both proteins were modified in their terminal extremity with a polyhistidine tag and co-expressed in the yeast P. pastoris. Proteins were purified in a mixture of lipids and detergent DDM using metal affinity chromatography followed by gel filtration. The crystal structure revealed that the pore of Kv1.2 is similar to that of prokaryotic K+ channels. In 2007, Long and co-workers crystallized a chimaeric voltage-dependent K+ channel in complex with lipids. The chimera

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was expressed in the yeast P. pastoris with a ten-histidine tag in its N-terminus and purified in a mixture of detergents and lipids using metal affinity chromatography followed by gel filtration. The complete structure at 2.4-Å resolution revealed the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules (15). The crystal structure of the human leukotriene C4 synthase in its apo and glutathione-complexed forms to 2.00- and 2.15-Å resolution, respectively, was also derived from heterologous expression in the yeast P. pastoris (16). The recombinant protein was purified using two affinity chromatography steps followed by a gel filtration in which the detergents used for membrane solubilization (Triton X-100 and sodium deoxycholate) were exchanged for DDM. The N-terminus hexahistidine tag used for purification has not been eliminated and is clearly visible in the structure. The same protein with a hexahistidine tag in the C-terminus was produced in the fission yeast S. pombe and provided a structure in complex with glutathione at 3.3-Å resolution (10). Structures derived from both expression systems are comparable. The structure of a MP from the same family, the human 5-lipoxygenase-activating protein (FLAP) modified with a polyhistidine tag in the C-terminus, was derived from the recombinant protein expressed in E. coli (12). This is the only structure of human PM derived from bacteria. Schertler and collaborators determined in 2007 the first structure of a recombinant GPCR transiently expressed in mammalian COS cells: the rhodopsin mutant N2C/D282C. The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases the thermal stability of rhodopsin. Recombinant rhodopsin was solubilized in DDM and purified using antibody resin. DDM was exchanged for C8E4 on gel filtration, and the recombinant protein was further purified using an ion-exchange column (19). The second structure of a recombinant GPCR, the human β2-adrenergic receptor, derives from recombinant proteins expressed in baculovirus-infected insect cells. In the first strategy, the human β2-adrenergic receptor was produced in a complex with an antibody fragment, solubilized in DDM and purified by sequential antibody and ligand affinity chromatography (18). A higher-resolution structure (2.4 Å) was obtained from an engineered human β2-adrenergic receptor fused in the middle of the third cytoplasmic loop to T4 lysozyme, solubilized in DDM and purified by sequential antibody and ligand affinity chromatography (11). Together with Tate, Schertler reported the 2.7-Å resolution structure of a mutant β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The turkey receptor was modified by limited mutagenesis to improve thermostability and

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expressed in baculovirus-infected insect cells. After solubilization in decylmaltoside, the recombinant protein was purified by affinity chromatography thanks to the hexahistidine tag added at its C-terminus, and decylmaltoside was exchanged for octylglucoside for crystallization (21). To obtain crystals allowing the structure determination at 1.9-Å resolution of acid-sensing ion channel 1 (ASIC), Jasti and collaborators (14) produced in baculovirus-infected Sf9 cells a mutant recombinant protein that possesses a deletion of 25 N-terminal and 64 C-terminal residues. Recombinant ASIC was purified in DDM using metal and size exclusion chromatography.

5. Conclusion This review presents an overview of the heterologous expression systems that allow overproduction of MPs for structural analysis. The two first structures of mammalian MPs derived from heterologous expression were solved in 2005. Since that time, nine new structures have been obtained from recombinant mammalian MPs heterologously expressed. From the 11 recombinant mammalian MP atomic structures obtained since 2005, 5 were derived from MPs expressed in yeast, 4 from MPs expressed in insect cells, 1 from mammalian cells and 1 from bacteria. These recent structures of mammalian MPs were obtained thanks to the use of recombinant proteins that enabled the exploration of mutants designed to improve crystal quality.

Acknowledgments I thank M. Bidet, O. Joubert and R. Nehmé for critical reading of the manuscript and the European Commission through the FP6 specific targeted research project Innovative Tools for Membrane Structural Proteomics (LSH-2003-1.1.0-1) that financially supported several authors of this volume for their research on heterologous expression systems, new surfactant molecules and MP stabilization. References 1. Liu J, Rost B (2001) Comparing function and structure between entire proteomes. Protein Sci 10:1970–1979 2. Lundstrom K (2006) Structural genomics for membrane proteins. Cell Mol Life Sci 63:2597–2607

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Heterologous Expression of Membrane Proteins for Structural Analysis 5. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647–655 6. Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346:967–989 7. Jidenko M, Nielsen RC, Sorensen TL et al (2005) Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 102: 11687–11691 8. Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltagedependent Shaker family K+ channel. Science 309:897–903 9. Junge F, Schneider B, Reckel S, Schwarz D, Dotsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol Life Sci 65:1729–1755 10. Ago H, Kanaoka Y, Irikura D et al (2007) Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis. Nature 448:609–612 11. Cherezov V, Rosenbaum DM, Hanson MA et al (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318: 1258–1265 12. Ferguson AD, McKeever BM, Xu S et al (2007) Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317:510–512 13. Hiroaki Y, Tani K, Kamegawa A et al (2006) Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol 355:628–639 14. Jasti J, Furukawa H, Gonzales EB, Gouaux E (2007) Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449:316–323 15. Long SB, Tao X, Campbell EB, MacKinnon R (2007) Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450:376–382 16. Martinez Molina D, Wetterholm A, Kohl A et al (2007) Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase. Nature 448:613–616 17. Pedersen BP, Buch-Pedersen MJ, Morth JP, Palmgren MG, Nissen P (2007) Crystal structure of the plasma membrane proton pump. Nature 450:1111–1114 18. Rasmussen SG, Choi HJ, Rosenbaum DM et al (2007) Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450:383–387

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Chapter 2 Production of Membrane Proteins in Escherichia coli and Lactococcus lactis Eric R. Geertsma and Bert Poolman Abstract As the equivalent to gatekeepers of the cell, membrane transport proteins perform a variety of critical functions. Progress on the functional and structural characterization of membrane proteins is slowed due to problems associated with their (heterologous) overexpression. Often, overexpression fails or leads to aggregated material from which the production of functionally refolded protein is challenging. It is still difficult to predict whether a given membrane protein can be overproduced in a functional competent state. As a result, the most straightforward strategy to set up an overexpression system is to screen a multitude of conditions, including the comparison of homologues, type and location of (affinity) tags, and distinct expression hosts. Here, we detail methodology to rapidly establish and optimize (membrane) protein expression in Escherichia coli and Lactococcus lactis. Key words: Arabinose promoter, Escherichia coli, folding indicator, GFP, Lactococcus lactis, membrane proteins, nisin-controlled expression, overexpression, PBAD

1. Introduction Although for the elementary steps in the biogenesis of membrane proteins, which are membrane targeting, membrane insertion, and assembly and folding, the crucial components are known (reviewed in (1)), our current understanding of the overall process and the factors involved are far from complete. Predicting whether a given membrane protein can be overproduced in a functional competent state is currently not possible, and bottlenecks have not been identified that could guide a rational troubleshooting strategy should overexpression fail. To date, membrane protein biogenesis has been mostly studied from the perspective of the known components involved in targeting, membrane insertion, and folding. In general, this is done using a limited number of

I. Mus-Veteau (ed.), Heterologous Expression of Membrane Proteins, Methods in Molecular Biology, vol. 601 DOI 10.1007/978-1-60761-344-2_2, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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small and simple model proteins, while systematic genomewide studies have indicated that overexpression of complex membrane proteins with multiple transmembrane segments is most challenging (2–4). Only recently efforts have been made to understand the complete process in the context of a cell by determining the physiological responses underlying (un)successful overproduction of (complex) membrane proteins (5,6). As a consequence, if one wishes to obtain structural and functional information on a certain membrane protein that is not abundant in any natural source, a convenient strategy is to screen a wide range of conditions. Established initial variables involve the screening of homologues (7,8), varying the type and location of affinity tags and domains (9,10), and screening multiple-expression hosts (11,12). In practice, for prokaryotes screening is restricted to several Escherichia coli strains, which offers far less variation than the screening of different species. The gram-positive bacterium Lactococcus lactis is an attractive alternative to E. coli and yeast-based membrane protein expression systems (13,14). Overexpression of membrane proteins in L. lactis leads mostly to well-folded, membrane-inserted material; inclusion bodies of aggregated material are rare. The lactococcal membrane is easily solubilized with a wide range of detergents. Growth of L. lactis proceeds rapidly at 30°C and does not require aeration. The nisin-inducible controlled expression (NICE) system (15) allows reproducible and modulatable expression from low to high levels. Previous difficulties associated with direct cloning in L. lactis (10) have been overcome by the high-throughput compatible vector backbone exchange (VBEx) procedure (16). As L. lactis is auxotrophic for multiple amino acids, adjustment of the lactococcal chemically defined medium (17) allows incorporation of labeled amino acid analogues into proteins (44, 45). Concerning membrane protein expression, there are five important differences between E. coli and L. lactis. First, the composition of the machinery involved in membrane protein insertion: L. lactis contains two paralogs of the Oxa/YidC/Alb family of membrane-inserted chaperones (18), whereas E. coli contains one (YidC). Lactococcus lactis does not contain homologues of the Sec translocon accessory proteins SecDF (19) and SecM (20). Finally, substantial differences in structure are observed between lactococcal and E. coli homologues of YidC and SecE (20,21). Second, the lipid composition of the membrane is very different: The major lipid component of the E. coli cytoplasmic membrane is phosphatidylethanolamine (PE), which is absent in L. lactis. Instead, anionic glycolipids and phosphoglycolipids are the major constituents in L. lactis (22). The third difference involves codon usage (23); L. lactis DNA is relatively AT rich (64% AT, compared to 50% AT in E. coli). The fourth difference is the absence of disulfide isomerase homologues in L. lactis (24), albeit that disulfides

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are readily formed either spontaneously or via an alternative route in secreted proteins (25–27). The final difference is that some proteins toxic to one host do not affect the physiology of the other host (C. Mulligan and E.R. Geertsma, March 2007). In addition, although fewer attempts have been made than in E. coli, overexpression of eukaryotic membrane proteins in L. lactis is relatively successful (28). 1.1. High-Throughput Cloning in E. coli and L. lactis

To establish an overexpression system rapidly, several homologues, types and location of affinity tags, and expression hosts need to be screened. Due to the large number of plasmids required for this strategy, it will be advantageous to avoid low-throughput techniques and implement efficient cloning procedures such as ligationindependent cloning (LIC) (29). LIC is less restricted in the design of the sequences flanking the genes than other high-throughput cloning techniques, like Gateway (30) or the univector plasmidfusion system (31). Therefore, the cloning-related sequences attached to the recombinant protein can be minimized, which might be advantageous for structural and functional analysis. The LIC procedure described here, outlined in Fig. 2.1a, involves linearization of the vector by a unique SwaI site in the middle of the LIC cassette. Using the 3¢ to 5¢ exonuclease activity of T4 DNA polymerase, single-stranded overhangs are generated. By performing the exonuclease treatment in the presence of dCTP (deoxycytidine triphosphate), a nucleoside not present in the 3¢ sequences adjacent to the SwaI site, the removal of bases halts once the first dCMP (deoxycytidine monophosphate) is encountered, thereby creating overhangs of a defined length. Complementary single-stranded overhangs are created in a similar way in the polymerase chain reaction (PCR) product to be cloned (i.e., exonuclease treatment in the presence of dGTP (deoxyguanosine triphosphate)). On mixing the T4 DNA polymerasetreated vector and PCR product, a stable heteroduplex is formed, and the mixture can be transformed with high efficiency to E. coli. The efficiencies for direct cloning in L. lactis are low, and LIC is virtually not possible. This bottleneck in cloning has been overcome by the VBEx procedure (16). Using VBEx, the initial LIC step can be performed with high efficiency in E. coli while the final expression vector that is generated can be kept devoid of alien E. coli elements. The VBEx procedure, outlined in Fig. 2.1b, relies on the bisection of a bona fide plasmid of the expression host into two parts, thereby separating the selection marker from the origin of replication. For L. lactis, pNZxLIC, a LICcompatible derivative of the well-established expression vector pNZ8048 (15) was accordingly bisected. The segment containing the origin of replication was fused to a sequence coding for an erythromycin selection marker, yielding plasmid pERL, which can be maintained in L. lactis. The other segment of pNZxLIC,

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Fig. 2.1. High-throughput cloning in recalcitrant bacteria using LIC and VBEx. (a) Outline of the LIC procedure. Gene X is amplified using primers holding LIC-specific overhangs. The plasmid is linearized by SwaI restriction in the LIC cassette. Singlestranded overhangs on the PCR product and vector are generated using T4 DNA polymerase. The complementary overhangs of PCR product and vector anneal on mixing. The resulting heteroduplex is transformed efficiently to Escherichia coli. (b) Outline of the VBEx strategy. After introduction of two distinct SfiI sites, the Lactococcus lactis expression vector pNZxLIC was bisected. Plasmid pERL received the pSH71 replicon, which was fused to the erythromycin marker. Plasmid pRExLIC received the chloramphenicol marker and LIC sequence, which were fused to the E. coli pBR322 replicon and β-lactamase marker. As these vectors are available, in practice the cloning involves the submission of vector pRExLIC to the LIC procedure (depicted in a). Subsequently, the VBEx procedure is applied: The pNZxLIC vector is restored by mixing pERL and pRExLICgene X, digestion with SfiI, ligation, and selection on the ability to replicate in L. lactis (the presence of the pSH71 replicon) in the presence of chloramphenicol. (c) The SfiI sites flanking both segments of the vectors yield different, nonpalindromic overhangs, thereby greatly reducing the number of possible ligation products. (Adapted from (16)).

containing the chloramphenicol resistance gene and LIC sequence, was fused to the backbone of an E. coli vector (containing an E. coli origin of replication and β-lactamase resistance gene). The resulting plasmid pRExLIC, allows the LIC manipulation to take place in E. coli.

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Rapid and facile regeneration of the L. lactis expression vector is enabled as the relevant segments of the pRExLIC and pERL vectors are flanked with distinct SfiI sites. DNA cleavage by SfiI generates a 3¢ overhang that can be composed of any combination of three nucleotides. The two SfiI sites used yield different, incompatible overhangs (Fig. 2.1c). Each segment of the pNZxLIC vector has unique selectable properties: (1) the ability to replicate a plasmid in L. lactis and (2) the resistance to chloramphenicol. Thus, on mixing a pRExLIC derivative and pERL, digestion by SfiI, followed by ligation and transformation to L. lactis in combination with selection of clones using chloramphenicol, the pNZxLIC derivative can be exclusively recovered with high efficiencies. DNA sequences from virtually all sequenced genomes are compatible with VBEx as SfiI sites are rare (16). Furthermore, genes containing internal SfiI sites are not necessarily excluded as 64 different 3¢ overhangs can be generated after SfiI digestion. Internal SfiI sites with 3¢ overhangs not matching those of the vector will not form a bottleneck in the procedure. 1.2. Optimization of Membrane Protein Overexpression

Following initial screening for expression, systematic optimization of the production conditions to increase the amount of well-folded protein inserted into the membrane is often required. As indicated, membrane protein biogenesis requires several additional steps and components beyond translation. Exceeding the capacity of the cell to process the nascent membrane protein correctly may reduce the final yield of well-folded material. This is especially true for E. coli as membrane protein production in this host is regularly accompanied by inclusion body formation. Adjustment of the expression rate is most easily done by varying the inducer concentration for well-tunable promoters, such as the E. coli PBAD (32) or the lactococcal PnisA (15). In addition, variation of the growth temperature during induction is an important parameter to affect the expression rate (33,34). While traditionally optimization of functional expression involved time-consuming steps such as the isolation of membrane vesicles and activity assays, this process has been greatly accelerated by the use of green fluorescent protein (GFP), fused to the C-terminus of the target protein, as folding reporter. Proper maturation of GFP in E. coli, leading to a fluorescent species that maintains its folded state during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), depends highly on the correct folding of its fusion partner (35–38). Consequently, only correctly folded target yields a fluorescent fusion protein that has a higher electrophoretic mobility than the misfolded fusion protein. Plain SDS-PAGE and subsequent immunoblotting allow simultaneous quantification of both the well-folded and aggregated protein produced (Fig. 2.2) (38). This facilitates the assignment of bottlenecks limiting the functional overexpression. For instance, the absence of the upper, nonfluorescent band suggests that transcription or

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Fig. 2.2. SDS-PAGE analysis of membrane protein GFP fusions expressed to different levels. Expression of the GFP fusion proteins was controlled by the PBAD, and Escherichia coli MC1061 cultures were induced with the percentage L-arabinose (w/v) indicated above the lanes. Upper panels represent the images obtained by in gel GFP fluorescence. Lower panels represent immunoblots of the same gels decorated with anti-His tag antibody. Black and white arrows indicate the positions of the aggregated nonfluorescent and well-folded fluorescent species of the GFP fusion proteins, respectively. (Adapted from (38)).

translation limits the functional expression, which can be resolved by increasing the inducer concentration. In contrast, the strong presence of the upper, nonfluorescent band suggests that the trajectory beyond transcription/translation is limiting. In addition to this application, the use of GFP fusions has additional advantages at the stages beyond membrane protein overexpression, such as the rapid screening of detergents for solubilization and stabilization (37,39).

2. Materials 2.1. LigationIndependent Cloning Procedure 2.1.1. Preparation of the DNA Insert

1. Autoclaved TlowE: 10 mM Tris-HCl, pH 7.5, 0.2 mM Na2EDTA (ethylenediaminetetraacetic acid). 2. Adhesive plate seals (e.g., plate seal AB-0580 from Thermo Scientific). 3. Phusion DNA polymerase and dedicated buffers (Finnzymes). 4. DNA gel extraction kit (e.g., the High Pure PCR Product Purification kit from Roche).

2.1.2. Preparation of the LIC Vector

1. LB-amp: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L of demineralized water. Sterilize by autoclaving. Once the medium is cooled to room temperature, add 1 mL of a filter-sterilized solution of 100 mg/mL ampicillin dissolved in water. 2. Plasmid isolation kit (e.g., the Qiaprep Spin Miniprep kit from Qiagen). 3. 10% (w/v) SDS solution. 4. Proteinase K solution: Dissolve 20 mg/mL proteinase K in 10 mM Tris-HCl, pH 8.0, 1 mM Na2-EDTA. Store aliquots at −20°C. 5. Phenol:chloroform:isoamyl alcohol (25:24:1) mixture. Phenol is extremely toxic; wear protective clothing and work in a fume hood.

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6. Chloroform, analytical grade. Chloroform is irritating and a carcinogen; wear protective clothing and work in a fume hood. 7. 96% EtOH, analytical grade, precooled at −20°C. 8. 3M Na-acetate, pH 5.3; adjust the pH with concentrated acetic acid. 9. 70% EtOH, analytical grade, precooled at −20°C. 10. SpeedVac (Savant). 11. SwaI and dedicated buffer. 12. Chemically competent E. coli MC1061. Protocols for the preparation of chemically competent E. coli can be found in (40). 13. LB-amp agar plates: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L of demineralized water and add 15 g agar. Sterilize by autoclaving. Once the medium is cooled to 50–60°C, add 1 mL of a filter-sterilized solution of 100 mg/ mL ampicillin dissolved in water; mix well and pour the medium in sterile Petri dishes. 2.1.3. T4 DNA Polymerase Treatment of Vector and Insert

1. T4 DNA polymerase and dedicated buffer. 2. 25 mM dCTP: Dilute a 100 mM dCTP stock in sterile Milli-Q. Store aliquots at −20°C. 3. 25 mM dGTP: Dilute a 100 mM dGTP stock in sterile Milli-Q. Store aliquots at −20°C. 4. Heat block.

2.1.4. Verifying Clones by Colony PCR

1. Autoclaved Milli-Q. 2. Sterile toothpicks. 3. 96-well PCR plates. 4. Aluminum sealing films (e.g., PCR-AS-200 from Axygen). 5. Taq DNA polymerase and dedicated buffer. 6. dNTPs: Make a stock solution containing 10 mM of each nucleotide. Store aliquots at −20°C. 7. Generic primers (see Note 1).

2.2. Vector Backbone Exchange Procedure 2.2.1. Preparation of pERL

1. GM17-ery: Dissolve 37.25 g of M17 broth (Oxoid) (41) in 1 L of demineralized water. Sterilize by autoclaving. Once the medium is cooled to room temperature, add 25 mL sterile 20% (w/v) glucose and 1 mL of a filter-sterilized solution of 5 mg/mL erythromycin dissolved in water. 2. Resuspension buffer: 10 mM Tris-HCl, pH 8.1, 10 mM Na2EDTA, 50 mM NaCl, and 20% (w/v) sucrose. Autoclave and store at 4°C. Supplement with 20 mg/mL lysozyme prior to use.

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2.2.2. Vector Backbone Exchange

1. SfiI and dedicated buffer. 2. 50 mM ATP stock: 50 mM Na2-ATP, 50 mM MgSO4, and 50 mM phosphate buffer, pH 7.0. Adjust the pH to 6.5–7.0 with NaOH. Store aliquots at −20°C. 3. T4 DNA ligase and dedicated buffer.

2.2.3. Electrotransformation of L. lactis

1. Sterile electroporation cuvets (2 mm gap between the electrodes). The cuvets can be reused several times. Clean the cuvets after use with 70% EtOH and wash extensively with Milli-Q. Allow the cuvets to dry and sterilize the cuvets by 20 min exposure to an ultraviolet (UV) source. Immediately close the cuvets with the supplied cap. 2. Electroporation device. 3. Rescue medium: Mix 1 volume of sterile M17 medium with 1 volume of sterile 1M sucrose, 1% (w/v) glucose, 40 mM MgCl2, 4 mM CaCl2. 4. SGM17-cam agar plates: Mix 37.25 g of M17 broth (Oxoid) and 15 g agar in 750 mL of demineralized water. Sterilize by autoclaving. Once the medium is cooled to about 60°C, add 250 mL sterile 2M sucrose, 25 mL 20% (w/v) glucose, and 1 mL of a filter-sterilized solution of 5 mg/mL chloramphenicol dissolved in EtOH. Mix well and pour the medium in sterile Petri dishes. 5. Parafilm.

2.3. Initial Characterization and Optimization of Protein Production 2.3.1. Cultivation and Induction of MP Overexpression in E. coli

1. Sterile 87% (w/v) glycerol. 2. Cryovials. 3. TB-amp: Dissolve 24 g tryptone, 48 g yeast extract, and 17.2 mL 87% (w/v) glycerol in 0.9 L of demineralized water and autoclave. In addition, autoclave 100 mL of 170 mM KH2PO4, 720 mM K2HPO4. Mix the solutions prior to use and add 1 mL of 100 mg/mL ampicillin. 4.

L-Arabinose:

Filter-sterilize a 20% (w/v) L-arabinose stock.

5. Screw-cap tubes compatible with the FastPrep-24 (e.g., screw-cap microtube 72.693 from Sarstedt). 2.3.2. Cultivation and Induction of MP Overexpression in L. lactis

1. GM17-cam: M17 medium supplemented with 0.5% (w/v) glucose and 5 mg/mL chloramphenicol after sterilization. 2. Nisin-containing supernatant of a L. lactis NZ9700 culture: Use a 1% (v/v) inoculum of a preculture of L. lactis NZ9700 on GM17 to inoculate 200 mL GM17 and incubate overnight at 30°C. Next day, pellet the cells, filtrate the supernatant using a 0.2-mm filter, and store the aliquots at −20°C.

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1. Glass beads with a diameter of about 0.1 mm (SigmaAldrich). 2. Disruption buffer: 50 mM phosphate buffer, pH 7.5, 10% (w/v) glycerol, and 1 mM MgSO4. Precool the solution on ice. Add 1 mM PMSF (phenylmethylsulfonyl fluoride) (from a 200 mM stock in EtOH (ethanol)) and 0.1 mg/mL DNase (deoxyribonuclease) prior to use. 3. FastPrep-24 device (MP Biomedicals). 4. 5X protein sample buffer: 120 mM Tris-HCl, pH 6.8, 50% (w/v) glycerol, 100 mM dithiothreitol (DTT), 2% (w/v) SDS, and 0.1% (w/v) bromophenol blue (see Note 2). Store aliquots at −20°C.

2.3.4. SDS-PAGE, In Gel GFP Fluorescence, and Western Blotting

1. SDS-PA gel: For a 10% running gel, mix 3.38 mL 30% acrylamide/bis solution (cross-linker ratio 37.5:1), 2.54 mL 1.5M Tris-HCl, pH 8.8, 4.15 mL Milli-Q, 100 mL 10% (w/v) SDS, 40 mL 10% (w/v) APS (ammonium persulfate), and 10 mL TEMED. For the stacking gel, mix 0.68 mL 30% acrylamide/bis solution (cross-linker ratio 37.5:1), 1.13 mL 0.5M Tris-HCl, pH 6.8, 2.25 mL Milli-Q, 50 mL 10% (w/v) SDS, 30 mL 10% (w/v) APS, and 5 mL TEMED (see Note 2). 2. Precision Plus Protein Dual Color standard (Bio-Rad). 3. LAS-3000 imaging system (Fujifilm). 4. Electroblotting and immunodetection system with primary antibody directed against a His-tag.

3. Methods 3.1. LigationIndependent Cloning Procedure 3.1.1. Preparation of the DNA Insert

1. Design two sets of LIC primers for each gene to be cloned. The nLIC and cLIC primer sets will finally produce proteins with an N- or C-terminal affinity tag, respectively. The LIC strategy requires the presence of dedicated 5¢ extensions on the primers (indicated in Table 2.1). In addition, the genespecific part of the primer should be designed so that it (1) does not contain the endogenous start or stop codon of the target gene; (2) is sufficiently long to anneal in a stable and selective way with the target DNA (in general, 20–25 nucleotides suffice; see Note 3). The primer should not contain strong secondary structure elements that could interfere with the PCR (see Note 4). Keep the total length of the

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Table 2.1 Primer extensions needed for ligation-independent cloning Primer type

5¢ Extensions of primers

nLIC forward

5¢ AT GGT GAG AAT TTA TAT TTT CAA GGT

nLIC reverse

5¢ T GGG AGG GTG GGA TTT TCA TTA

cLIC forward

5¢ ATG GGT GGT GGA TTT GCT

cLIC reverse

5¢ TTG GAA GTA TAA ATT TTC

primers below 50 residues to ensure sufficient quality and cost-efficiency. 2. For ordering, select the lowest synthesis scale offered by the supplier and mere desalting as the purification step. For large sets (>48 primers), order the primers in a 96-well plate. 3. On delivery, spin down the material and dissolve the primers to 100 mM in TlowE and make a substock of 5 mM. Use a transparent sticker to seal the plates. The 100 mM stock can be stored for several months at −20°C. Store the 5 mM stock at 4°C. Spin down the material before use. 4. PCR the desired open reading frames using the Phusion DNA polymerase, which combines high fidelity with high production (see Notes 5 and 6). Prepare a 50 mL reaction mix according to the manufacturer’s protocol and add the polymerase just prior to the start of the reaction. Place the sample in a PCR machine preheated to 98°C and start the reaction. A good starting point for a touchdown PCR program (see Note 7) is a. 60 s at 98°C. b. 10 s at 98°C. c. 15 s at 58.5°C (decrease 0.5°C per cycle). d. XX s at 72°C (15–30 s/kb). Repeat steps 4a–4d 14 times. e. 10 s at 98°C. f. 15 s at 51°C. g. XX s at 72°C (15–30 s/kb). Repeat steps 4e–4g 14 times. h. 300 s at 72°C. i. Forever at 10°C. Once the PCR is finished, add DNA loading buffer to the sample and analyze all the material by TAE (Tris-acetate-EDTA) gel electrophoresis (see Note 8). Gel purify the band using a

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Table 2.2 Basic and additional LIC vectors Vector name

Protein sequence

Protein sequence after TEV protease cleavage

Expression host

pBADnLIC

M-His10-G-TEV site-protein

G protein

Escherichia coli

pBADcLIC

MGGGFA-protein-TEV site-His10

MGGGFA-proteinENLYFQ

E. coli

pBADcLIC-GFP

MGGGFA-protein-TEV site-GFP-His10

MGGGFA-proteinENLYFQ

E. coli

pBAD-OmpA-nLIC

M-ssOmpAa-His10-G-TEV site-protein

G-protein

E. coli

pREnLIC

M-His10-G-TEV site-protein

G-protein

Lactococcus lactis

pREcLIC

MGGGFA-protein-TEV site-His10

MGGGFA-proteinENLYFQ

L. lactis

pREcLIC-GFP

MGGGFA-protein-TEV site-GFP-His10

MGGGFA-proteinENLYFQ

L. lactis

pRE-USP45-nLIC

M-ssUSP45b-His10-G-TEV site-protein

G-protein

L. lactis

a ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane or to replace the signal sequence of the protein of interest with the ssOmpA b ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45 -nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane or to replace the signal sequence of the protein of interest with the ssUSP4

commercial gel extraction kit according to the manufacturer’s protocol. Determine the DNA concentration spectrophotometrically (see Note 9). Store the material at −20°C. 3.1.2. Preparation of the LIC Vector

1. Inoculate approximately 20 mL LB-amp with E. coli MC1061 containing one of the pBAD- or pRE-derived LIC vectors (Table 2.2). Cultivate overnight at 37°C with vigorous shaking. 2. Isolate the plasmid using a commercial plasmid isolation kit and elute the plasmid with TlowE. Remove remaining proteins by combining a proteinase K treatment with a phenol:chloroform extraction as described in steps 3–5. 3. Combine several fractions and adjust the volume to 300 mL. Supplement the sample with SDS to a final concentration of 0.5% and add proteinase K to a final concentration of 40 mg/mL. Incubate for 30 min at 60°C to digest remaining proteins.

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4. In a fume hood, add 1 volume of phenol:chloroform and vortex shortly. After 2 min, the sample is vortexed again and centrifuged for 2 min at full speed in a table centrifuge. Collect most of the top phase without disturbing the (white) interface and transfer to a new cup. Add 1 volume of chloroform and mix by vortexing. Centrifuge for 2 min at full speed in a table centrifuge and transfer the top phase without disturbing the interface to a new cup. 5. Add 2.5 volume of ice-cold 96% EtOH and 0.1 volume of 3M Na-acetate, pH 5.3. Vortex the sample and precipitate the DNA for 30 min at −80°C. Next, centrifuge the sample at 4°C for 15 min at full speed in a table centrifuge. Remove the supernatant and add 0.9 mL of ice-cold 70% EtOH. Centrifuge 5 min at full speed at room temperature in a table centrifuge. Remove the supernatant and dry the pellets shortly in a SpeedVac. Dissolve the pellet in 20 mL TlowE, determine the DNA concentration, and verify the integrity of the plasmid by restriction analysis. 6. Take 5 mg plasmid and digest the plasmid in a total volume of 40 mL containing 20 U SwaI according to the manufacturer’s protocol (see Note 10). After 3 h, supplement the reaction with 10 U SwaI and extend the reaction for another 3 h. Analyze the full sample on 0.8% (w/v) agarose TAE gel using combs with wide wells. Isolate the linearized vector using a gel purification kit and determine the concentration. Store the material at −20°C. 7. To test the completeness of the digestion, transform 15 ng of the linearized plasmid to chemically competent E. coli MC1061 according to standard procedures. After the transformation procedure and 1 h incubation at 37°C with LB medium, sediment the cells by centrifugation (7,600g, 5 min), remove most of the supernatant, and plate the resuspended material on LB-amp agar plates. After overnight incubation, the colony count should be low ( 2). 2. Dilute the 30-mL preculture in 300 mL minimal medium (270 mL YNB + 30 mL 10X D + 3 mL 100X AA) in a 2-L autoclaved Erlenmeyer flask. 3. Keep at 30°C under shaking at 200 rpm. Let yeast grow until 2 OD. Afternoon 1. Put 500 mL of rich medium (450 mL YEP + 50 mL of 10X D solution) in each of the twelve 2-L Erlenmeyer flasks prepared for culture. 2. Inoculate each Erlenmeyer flask with 25 mL of your preculture (check your culture for any bacterial contamination by microscopic observation). Thus, your culture starts at 0.1 OD. 3. Change your incubator’s temperature to 18°C. Day 3: 1. Check OD600 in the morning. Yeast should have reached an OD600 around 2. 2. Check microscopically to determine if your culture has been contaminated. 3. At 24 h postincubation, yeasts should have reached an OD600 about 6–7. 4. Harvest the cells by centrifugation for 10 min at 3000 g at +4°C. 5. The wet pellet in these conditions should weigh around 60 g (which indicates 10 g/L culture). 6. Resuspend the pellet first with cold water. 7. Centrifuge 10 min at 3000 g at 4°C. Discard the supernatant. 8. Wash with ice-cold buffer A (1X) containing 50 mM Tris-HCl, 500 mM NaCl, pH 7.4, supplemented with protease inhibitors: 1 mM PMSF, 4 mM benzamidine hydrochloride, 2.5 mM EDTA, pH 8.

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9. Centrifuge again 10 min at 3000 g at 4°C. Discard the supernatant. 10. Freeze the pellet at −80°C until next use. 3.4. Yeast Membrane Fraction Preparation

1. Make 200 mL of buffer A (1X) by mixing 100 mL of ice-cold buffer A (2X) with 1 mL PMSF (200X), 1 mL EDTA (200X), 1 mL benzamidine hydrochloride (200X) and complete to 200 mL with ice-cold Milli-Q water. 2. Thaw yeast pellet by adding 3 volumes of this freshly prepared ice-cold buffer A supplemented with protease inhibitors. 3. In a 50-mL Falcon tube, put 17.5 mL of yeast solution and 2.5 mL of glass beads (see Note 6). 4. Grind yeast by vortexing at 2,000 rpm at 4°C for 15 min. This is the optimal time, as shown in Fig. 6.1 using a Heidolph multireax device. 5. With a pipet, separate the supernatant from the beads and centrifuge at 3,000 g for 10 min to eliminate unbroken cells, debris, and nuclei. Here you, can estimate the grinding’s efficiency by measuring the ratio between the starting and the remaining volume of pellet (see Note 7). 6. The supernatant is then centrifuged at 18,000g for 1 h at 4°C. 7. The supernatant is discarded.

Fig. 6.1. Saccharomyces cerevisiae grinding with glass beads. Each 50-mL Falcon tube corresponding to 1 L of culture was vortex shaken at maximum speed (2000 rpm) during the indicated time (1–60 min). Membrane proteins were prepared as indicated in the protocol, and the total amount of material was estimated by the Bradford method.

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8. This pellet containing yeast plasma membrane is washed twice in the same buffer without EDTA. Again, centrifuge at 18,000g for 1 h at +4°C. 9. Make 200 mL of buffer B 1X by mixing 100 mL of ice-cold buffer B (2X) with 1 mL PMSF (200X) and 1 mL benzamidine hydrochloride (200X) and complete to 200 mL with ice-cold water. 10. Finally, the pellet is resuspended in buffer B plus protease inhibitors without EDTA. It is necessary to remove EDTA before solubilizing membrane fraction for purification on a Ni-NTA column. 11. Using DC Bio-Rad assay reagents and bovine serum albumin (BSA) at concentrations ranging from 2 to 20 mg/mL as a standard, quantify total MPs. 3.5. SDS Polyacrylamide Gel Electrophoresis

Tables to help you make separating and stacking gels with different acrylamide concentrations are easily available (see Tables 6.1 and 6.2). These instructions assume the use of the Bio-Rad MiniProtean gel system. 1. Glass plates to be used should be well cleaned and extensively rinsed with distilled water and ethanol. 2. Prepare a 0.75-mm thick, 8% separating gel. See Table 6.1 for gel composition. Wear gloves because acrylamide is neurotoxic and carcinogenic when unpolymerized. 3. Pour the gel, leaving space (1 cm below comb teeth) for a stacking gel, and overlay with isobutanol or isopropanol (30–50 mL). Allow the gel to polymerize for at least 10 min. Polymerization time depends on room temperature. 4. Pour off the isobutanol or isopropanol and rinse twice with water. 5. Prepare the stacking gel. See Table 6.2 for composition. Pour the gel and immediately insert the comb. Wait few minutes to polymerize before removing the comb. 6. Prepare the running buffer by diluting 200 mL of the 5X buffer with 800 mL of water. Mix before use. 7. Once the stacking gel has set, carefully remove the comb and wash wells with water using pipet (see Note 8). 8. Put the gel in the electrophoresis unit. 9. Mix 15 mL of your sample with 5 mL of the 4X sample buffer. 10. Screen heating time necessary to denature your protein without aggregating it. We tested 0, 2, 5, 10, and 15 min. This step depends on your protein (see Fig. 6.2).

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Table 6.1 Solutions for preparing resolving gels for Tris-glycine SDS-PAGE (Modified from Harlow and Lane 1988) Component volumes (mL) per gel mold volume (mL) of Solution components

5

10

15

20

25

30

40

50

H2O

2.6

5.3

7.9

10.6

13.2

15.9

21.2

26.5

30% acrylamide mix

1.0

2.0

3.0

4.0

5.0

6.0

8.0

10.0

1.5M Tris-HCl (pH 8.8)

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

10% SDS

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

10% ammonium persulfate

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

TEMED

0.004

0.008

0.012

0.016

0.02

0.024

0.032

0.04

H2O

2.3

4.6

6.9

9.3

11.5

13.9

18.5

23.2

30% Acrylamide mix

1.3

2.7

4.0

5.3

6.7

8.0

10.7

13.3

1.5M Tris-HCl (pH 8.8)

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

10% SDS

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

10% ammonium persulfate

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

TEMED

0.003

0.006

0.009

0.012

0.015

0.018

0.024

0.03

H2O

1.9

4.0

5.9

7.9

9.9

11.9

15.9

19.8

30% acrylamide mix

1.7

3.3

5.0

6.7

8.3

10.0

13.3

16.7

1.5M Tris-HCl (pH 8.8)

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

10% SDS

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

10% ammonium persulfate

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

TEMED

0.002

0.004

0.006

0.008

0.01

0.012

0.016

0.02

H2O

1.6

3.3

4.9

6.6

8.2

9.9

13.2

16.5

30% acrylamide mix

2.0

4.0

6.0

8.0

10.0

12.0

16.0

20.0

1.5M Tris-HCl (pH 8.8)

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

10% SDS

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

10% ammonium persulfate

005

0.1

0.15

0.2

0.25

0.3

0.4

0.5

TEMED

0.002

0.004

0.006

0.008

0.01

0.012

0.016

0.02

6%

8%

10%

12%

15% (continued)

97

Table 6.1 (continued) Component volumes (mL) per gel mold volume (mL) of Solution components

5

10

15

20

25

30

40

50

H2O

1.1

2.3

3.4

4.6

5.7

6.9

9.2

11.5

30% acrylamide mix

2.5

5.0

7.5

10.0

12.5

15.0

20.0

25.0

1.5M Tris-HCl (pH 8.8)

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

10% SDS

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

10% ammonium persulfate

0.05

0.1

0.15

0.2

0.25

0.3

0.4

0.5

TEMED

0.002

0.004

0.006

0.008

0.01

0.012

0.016

0.02

Table 6.2 Solutions for preparing 5% stacking gels for Tris-glycine SDS-PAGE (Modified from Harlow and Lane 1988) Component volumes (mL) per gel mold volume (mL) of Solution components

1

2

3

4

5

6

8

10

H2O

0.68

1.4

2.1

2.7

3.4

4.1

5.5

6.8

30% Acrylamide mix

0.17

0.33

0.5

0.67

0.83

1.0

1.3

1.7

1.0M Tris-HCl (pH 6.8)

0.13

0.25

0.38

0.5

0.63

0.75

1.0

1.25

10% SDS

0.01

0.02

0.03

0.04

0.05

0.06

0.08

0.1

TEMED

0.001

0.002

0.003

0.004

0.005

0.006

0.008

0.01

Fig. 6.2. Anti-HA Western blot. Each sample contains an equivalent amount of membrane proteins (expressing human receptor Smoothened, hSmo). Loading buffer (4X) was added, and then each sample was boiled for the indicated time (lanes 1–4; 0–15 min). The band corresponding to the hSmo disappears when the sample is boiled, probably due to aggregation.

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11. Load the samples into the wells with a Hamilton syringe or a pipet using gel loading tips. Load also 3 mL of prestained molecular weight markers. 12. If using Bio-Rad’s patented sample loading guide, place it between the two gels in the electrode assembly. 13. Use the sample loading guide to locate the sample wells. Insert the Hamilton syringe or pipet tip into the slots of the guide and fill the corresponding wells (see Note 9). 14. Remove the sample loading guide. 15. Place the lid on the minitank. Make sure to align the color-coded banana plugs and jacks. The correct orientation is made by matching the jacks on the lid with the banana plugs on the electrode assembly. A stop on the lid prevents incorrect orientation. 16. Insert the electrical leads into a suitable power supply with the proper polarity. 17. Constant 100 V is recommended for SDS polyacrylamide gel electrophoresis (SDS-PAGE). Run time is around 1 h 30 min. Time depends on the molecular weight of your protein. 18. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads. 19. Remove the tank lid and carefully lift out the inner chamber assembly. Pour off and discard the running buffer (see Note 10). 20. Open the cams of the clamping frame. Pull the electrode assembly out of the clamping frame and remove the gel cassette sandwiches. 21. Remove the gels from the gel cassette sandwich by gently separating the two plates of the gel cassette. The green, wedge-shaped, plastic gel releaser may be used to help pry the glass plates apart. 22. Remove the gel by floating it off the glass plate by inverting the gel and plate under fixative (for silver staining, for instance) or transfer solution (proceed to Section 6.3.6), agitating gently until the gel separates from the plate. 3.6. Western Blot

These directions for the Western blot assume the use of the BioRad Mini Trans-Blot electrophoretic transfer cell. 1. Prepare the 1X transfer buffer just before use by diluting 100 mL of the 10X transfer buffer in 700 mL Milli-Q water and add 200 mL ethanol (to have a final concentration of 20%). 2. Cut the membrane and the filter paper to the dimensions of the gel. Always wear gloves when handling membranes to prevent contamination. Soak the membrane, filter paper, and fiber pads in transfer buffer 15 min before blotting. 3. Prepare the gel sandwich.

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4. Place the cassette, with the black side down, in a recipient containing 1X transfer buffer. 5. Place one prewetted fiber pad on the gray side of the cassette. 6. Place a sheet of filter paper on the fiber pad. 7. Place the gel on the filter paper. 8. Place the prewetted membrane on the gel. Remove any air bubbles between gel and membrane by rolling a 5-mL tube on the membrane. 9. Complete the sandwich by placing a piece of filter paper on the membrane. 10. Add the last fiber pad. 11. Close the cassette firmly, being careful not to move the gel and filter paper sandwich. 12. Lock the cassette closed with the white latch. 13. Place the cassette into the transfer tank such that the nitrocellulose membrane is between the gel and the anode. It is vitally important to ensure this orientation or the proteins will be lost from the gel into the buffer rather than transferred to the nitrocellulose. 14. Add the frozen Bio-Ice cooling unit. Place in tank and completely fill the tank with buffer. 15. Put on the lid, plug the cables into the power supply, and run the blot under constant voltage of 100 V for 1 h (high-intensity field ~ 350 mA). 16. On completion of the run, disassemble the blotting sandwich and remove the membrane for development. Clean the cell, fiber pads, and cassettes with laboratory detergent and rinse well with deionized water. 17. The colored molecular weight markers should be clearly visible on the membrane. 18. To check whether your transfer has been correctly done, rinse the blot for 2 min with 0.1% of amidoblack solution to stain the transferred bands. 19. Wash twice with TBS-T buffer to remove amidoblack. 3.6.1. Classical Antibody Incubation Method (see Fig. 6.3, Lane 2)

1. The nitrocellulose is then incubated in 50 mL blocking buffer for 1 h at room temperature on a rocking platform. 2. The blocking buffer is discarded, and the membrane is placed in a plastic bag containing 5 mL of blocking buffer supplemented with the primary antibody (here 1:20 dilution of anti-HA antibodies) overnight at 4°C on a rotating wheel. 3. The primary antibody is then removed, and the membrane is washed three times for 10 min each with 50 mL blocking buffer.

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Fig. 6.3. Antibody incubation methods. After transfer, the nitrocellulose membrane was cut; the two methods were tested: With the classical method, exposing the blot for 20 s was sufficient to have the signal corresponding to hSmo, whereas with SNAP method, time exposure was longer (2 min) to obtain the same signal. Lane 1: membrane fraction of untransformed yeast. Lane 2: membrane fraction expressing hSmo detected using classical method. Lane 3: membrane fraction expressing hSmo detected with SNAP i.d method.

4. The membrane is placed in a plastic bag containing 5 mL of blocking buffer supplemented with the convenient secondary antibody at the dilution given by the manufacturer (here, it is the secondary polyclonal antimouse immunoglobulin antibody used as a 1:5,000-fold dilution). Place the bag on a rotating wheel for 2 h at 4°C (see Note 11). 5. The secondary antibody is discarded, and the membrane is washed twice for 10 min each with blocking buffer and a third time with TBS-T buffer (without milk). 3.6.2. Rapid Antibody Incubation Method (see Fig. 6.3, Lane 3)

Millipore has introduced a new method to optimize blotting conditions in record time for maximum results. Unlike conventional Western blotting, for which diffusion is the primary means of reagent transport, the SNAP i.d Protein Detection System applies a vacuum to actively drive reagents through the membrane. This novel method, compatible with all standard membranes, reduces incubation time and optimizes your blot in 30 min from blocking to revelation steps. This method requires a small amount of nonfat dry milk (as low as 0.2% w/v in our case for blocking, antibody dilution, or washing steps). In this method, time exposure is longer than in the classical method.

3.7. Expression Level as a Function of OD600

On the following, we analyzed the expression level of hSmo as a function of yeast cell density. 1. Measure yeast OD by diluting culture to 1:10 in a 2-mL spectrophotometer cuvette and reading the absorbance at 600 nm. 2. Stop culture at OD600 of 3, 5, 10, 15, and 20. 3. Prepare membrane fraction of each culture condition.

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4. Quantify protein using the Bradford method using a BioRad kit. 5. Protein expression in the membrane fraction increases and reaches a maximum around OD 7; therefore, we choose to cultivate to 7 OD (see Fig. 6.4). 3.8. Expression Level as a Function of Air-to-Medium Ratio

We studied the effect of culture Erlenmeyer air-to-medium ratio on the protein expression level. 1. In a 2 L-Erlenmeyer flask, pour 200, 300, 400, or 500 mL YEP medium. The air-to-medium ratio is therefore 10:1, 7:1, 5:1, and 4:1, respectively. 2. Culture is performed at 18°C under shaking at 200 rpm. 3. Let yeast grow until about 7 OD600. 4. Prepare membrane fraction for each culture condition. 5. Quantify protein using the Bradford method using a Bio-Rad kit. 6. As shown on the Western blot in Fig. 6.5, the expression level was higher when yeasts were grown with the highest oxygenation condition.

Fig. 6.4. Constitutive expression of hPtc in Saccharomyces cerevisiae plasma membrane fraction as a function of the yeast optical density (OD). Western blot revealed with an anti-HA antibody; 40 mg of protein were loaded in each lane.

Fig. 6.5. Constitutive expression of hSmo in Saccharomyces cerevisiae plasma membrane fraction as a function of the air-to-medium ratio in the culture flask. Western blot revealed with anti-HA antibodies. Expression yield was better when air volume in the culture flask was higher.

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4. Notes 1. Anti-HA antibody is stored at +4°C by addition of 0.01% final concentration sodium azide, conveniently done by dilution from a 10% stock solution. Azide is highly toxic; therefore, it should be cautiously handled. 2. To pellet the cells, 5 s are sufficient. 3. Usually, 1-3 µL should be sufficient. 4. DTT is instable in solution. It should be prepared, immediately aliquoted, and stored at −20°C. 5. Try to get as many cells as possible but do not worry about keeping every last cell. You should easily get 80–90% of cells. 6. This repartition seems to be optimal. Exceeding these volumes may alter vortexing; therefore, grinding will not be efficient. 7. To estimate grinding’s efficiency, you can compare the volume of the yeast pellet before and after grinding if you are using the 50-mL Falcon tubes. If not, weigh the pellet before and after grinding. Generally, this ratio is around 50–60%. 8. Remove the comb slowly and vertically to avoid damaging the wells. 9. Load samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the syringe needle or pipet tip. 10. Always pour off the buffer before opening the cams to avoid spilling the buffer. 11. You can try to reduce time by incubating the membrane with the secondary antibody for 1 h at room temperature.

Acknowledgment This work was supported by the European Community Specific Target Research Project grant FP6-2003-LifeSciHealth, “Innovative Tools for Membrane Structural Proteomics.” References 1. Orlean P (1997) Biogenesis of yeast wall and surface components, in The molecular and cellular biology of the yeast Saccharomyces cerevisiae: cell cycle and cell biology (Pringle, J.R., Broach, J.R., Jones, E.W. eds.), Cold Spring Harbor Laboratories, NY, pp 229–362

2. Lee AG (2004) How lipids affect the activities of integral membrane proteins. Biochim Biophys Acta 1666:62–87 3. Jidenko M, Nielsen RC, Sorensen TL, Moller JV, le Maire M, Nissen P, Jaxel C (2005) Crystallization of a mammalian membrane

Heterologous Expression of Human Membrane Receptors in the Yeast protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 102: 11687–11691 4. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647–655 5. Joubert O, De Rivoyre M, Mus-Veteau I, Nehmé R, Bidet M (2008) A strain of S. cerevisiae able to produce the human form of Patched, receptor of Sonic Hedgehog. (CNRS, ed) Patent number 08/06125, France 6. De Rivoyre M, Bonino F, Ruel L, Bidet M, Therond P, Mus-Veteau I (2005) Human

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receptor Smoothened, a mediator of Hedgehog signalling, expressed in its native conformation in yeast. FEBS Lett 579: 1529–1533 7. Figler RA, Omote H, Nakamoto RK, Al-Shawi MK (2000) Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Arch Biochem Biophys 376: 34–46 8. Sambrook J, Fritsch EF, Maniatis T (ed.) (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press, NY.

Chapter 7 Mammalian Membrane Protein Expression in Baculovirus-Infected Insect Cells Céline Trometer and Pierre Falson Abstract A system of expression of mammalian membrane proteins in baculovirus-infected insect cells is described, allowing analytical or preparative production in the milligram range of such type of proteins. This is illustrated by the setup of the expression system of the human breast cancer resistance protein (BCRP), which is a homodimeric multidrug ABC (adenosine triphosphate-binding cassette) transporter. The system used is Bac to Bac™, which allows generation of a viral genome that includes the protein of interest by using a shuttle plasmid and a bacterial host carrying the bacmid. In the present case, the use of a molecular chaperon, ninaA, is illustrated, co-expressed with BCRP by using a plasmid allowing the expression of two proteins, pFastBac Dual. The method is detailed to allow the expression of such proteins and membrane protein in general. Key words: ABC transporters, baculovirus, heterologous expression, insect cells, mammalian membrane proteins

1. Introduction Producing membrane proteins from higher eukaryotes (e.g., mammalian) in the milligram range and at the same time ensuring correct folding and full activity of the expressed protein remains the main bottleneck in obtaining structural information at high resolution of such membrane proteins. One main reason for this is that most of these membrane proteins require a specific maturation process to be fully active: They are produced in the endoplasmic reticulum and are then targeted to a given membrane through a process involving glycosylation (1) and association with given lipids (2), both steps achieved in the endoplasmic reticulum and Golgi apparatus. In that quest, to date the baculovirus-infected cells has been one of the most versatile expression systems, ensuring quantity and quality of membrane protein production while I. Mus-Veteau (ed.), Heterologous Expression of Membrane Proteins, Methods in Molecular Biology, vol. 601 DOI 10.1007/978-1-60761-344-2_7, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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remaining easy to handle and to scale up. Indeed, a large number of soluble and several membrane-bound proteins have been successfully produced using this system (3). Baculoviruses have been known for a long time, with early reports describing the infection of Chinese silkworms; these viruses infect only larval lepidopterans. Baculoviruses are enveloped, double-stranded DNA viruses of 80–180 kbp with rodshaped nucleocapsids. They exist as budded and occluded forms, the latter consisting of multiple nucleocapsids embedded in a polyhedrin (PH) matrix. The most extensively studied baculovirus strain is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Baculovirus gene expression is divided into two main phases, which precede (early phase) or follow (late phase) viral DNA replication. During the late phase, some hyperexpressed genes, such as those coding for p10 and polyhedron, remain transcribed at high levels. Such a type of promoters is used for protein expression, although in some cases those of early genes are used (4). The baculovirus expression vector system (BEVS) was initiated by Smith and Summers in 1982 (5). It is based on replacement of a late, non-essential, viral gene coding for the PH with a gene of interest. Most of the transfer vectors use either early (Ie1) or late (p10, pPH) promoters. Based on linearized vectors, the BEVS allows rapid cloning (1 week) and expression (2 weeks) of recombinant proteins in insect cells of lepidopterans such as the fall armyworm (Spodoptera frugiperda: Sf9, Sf21) or the cabbage looper (Trichoplusia ni, Hi5). The most popular BEVS are Bac to Bac™ and BaculoDirect™ (Invitrogen); flashBAC™/BacMagic (EMD/ OET/Nextgen); and BacPAK6/BaculoGold (BD Biosciences/ Clonetech). In each case, the system involves a bacterial-type plasmid including sequences of recombination; between them, the gene of interest is inserted and placed under the control of an early or late promoter. Insertion of the gene into the viral genome is carried out by recombination using bacteria containing the viral genome. After insertion, the viral genome is prepared in large extent and used to transfect the insect cells. Optimization of expression involves the time of expression (24–96 h postinfection, hpi), temperature (20–28°C) and type of culture medium. The overall process of expression is rather simple to set up and to scale up from plates to large volumes of liquid culture. One limitation concerns the glycosylation of proteins, which is distinct from that of mammalian cells; while the latter tends to glycosylate to a large extent, with complex N-glycans including mannose, N-acetyl glucosamine, galactose and at the end, sialic acid, insect cells glycosylate poorly and essentially with mannose (3). This problem can be partially solved by using genetically adapted cells, such as Mimic cells (Invitrogen), in which human glycosyltransferases have been introduced (6). A second limitation

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in the use of insect cells concerns their lipid composition, which differs significantly from that of mammalian cells; notably, the level of cholesterol is lower (7). Again, a solution can be found by adding cholesterol to the culture media (8). The present chapter details the experimental procedure to produce a membrane protein, here the human breast cancer resistance protein (BCRP), by using the Bac to Bac baculovirus-based expression system. BCRP belongs to the adenosine triphosphate (ATP)-binding cassette transporter family (9). It was initially identified for the resistance it confers to anticancer drugs (10, 11). The protein is addressed to the plasma membrane and organized as a homodimer. Each monomer is made of 655 residues that form an intracellular nucleotide-binding domain, a six-span membrane domain and a short extracellular domain. Expression of BCRP has been achieved in baculovirus-infected cells using Sf9 (12) or Hi5 (13, 14) cell lines, the latter producing a higher level of protein, but heterogeneously. We show that the use of a biological chaperon, NinaA (15, 16), can prevent this problem and can help produce more membrane protein.

2. Materials The material used for human BCRP expression via baculovirusinfected cells described here is provided from Invitrogen as included in the Bac to Bac expression kit. 2.1. Proteins

1. The gene coding for NinaA (membrane protein chaperon, UniProtKB/Swiss-Prot:P15425) was provided by Dr. Bertrand Mollereau, ENS Lyon. 2. The gene coding for human ABCG2 (UniProtKB/SwissProt:Q9UNQ0) was from a pTriEx construct described in (14).

2.2. Lab Equipment

1. All the material needed for molecular biology experiments. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots are carried out using the MiniProtean 3 apparatus and related devices from Bio-Rad. 3. Transfection and (solid and liquid) culture of insect cells are achieved in a class I type room (see Note 1) equipped with a Steril Bio Ban 48, an incubator Heraeus BK6160 with a H + P Biomag Biomodule 40B, a microscope Olympus CKX31 and a low-speed centrifuge handling 15-/30-mL Falcon-type tubes. 4. Six-well, T-25, T-75 and T-225 plates (Falcon) are used for adherent cell cultures. Liquid cultures of 250, 500, and 1,000 mL are carried out in spinners (Bellco).

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2.3. Cells

1. Insect cell line Sf9, High Five™, or Mimic™ Sf9 (not used here but useful to achieve full glycosylation if necessary). 2. MAX Efficiency® DH10Bac™ chemically competent Escherichia coli (Invitrogen) that contains a baculovirus shuttle vector (bacmid, bMON14272, 136 kb) and a helper plasmid, coding for a transposase, allowing recombination between the bacmid and the pFastBac™ Dual construct. 3. One Shot® TOP10 chemically competent E. coli or equivalent to generate the recombinant plasmid of interest.

2.4. Media

1. Insect cells are grown in Sf-900 II SFM or Express Five® SFM (both from Invitrogen) as appropriate. Both media contain 0.1% of the surfactant F68 pluronic acid and are supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. Express Five is supplemented with 2 mM glutamic acid. Media are stored for long term at 4°C. 2. Bacteria are grown in Luria-Bertani medium: 10 g/L Bactotryptone, 5 g/L Bacto-yeast extract, 10 g/L NaCl, supplemented with 15 g/L agar for plates. Autoclave 20 min at 120°C. Before use add antibiotics depending on the strain used, either 100 mg/L ampicillin (see Note 2) for TOP 10 or 50 mg/L kanamycin, 7 mg/L gentamicin, 10 mg/L tetracycline, 40 mg/mL IPTG (isopropyl-β-d-galactopyranoside) and 100 mg/ mL X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) for DH10Bac transformed with pFastBac Dual plasmid. Store without antibiotics at room temperature.

2.5. Transfection

1. Reagent: Cellfectin® reagent (Invitrogen, included in the Bac to Bac kit) is a 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIIItetrapalmitylspermine (TM-TPS) and dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water. Store at 4°C. 2. Medium: Unsupplemented Grace’s insect cell culture medium (Invitrogen). Store for long term at −20°C and short term (1 week) at 4°C. Note that Grace’s insect cell culture medium should not contain supplements or FBS as their protein content will interfere with the Cellfectin Reagent, inhibiting the transfection.

2.6. Molecular Biology

1. The plasmid used here is the pFastBac Dual vector (Invitrogen, included in the Bac to Bac kit), which contains two multiple cloning sites, allowing expression of two genes, controlled by (late) promoters of PH and p10.

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2. The kits for small- (3–10 mg) and medium- (50–100 mg) scale plasmid (5–10 kbp) DNA preparations are NucleoSpin™ Plasmid and Nucleobond® PC100 from Macherey-Nagel. 3. The kit for large DNA preparation (Bacmid, 150 kbp) is PureLink™ HiPure Plasmid Midiprep from Invitrogen. 4. Accuprime Taq DNA polymerase used for polymerase chain reaction (PCR) was from Invitrogen. Other restriction and modifying enzymes were from New England Biolabs. 2.7. SDS-PAGE

1. Prepare stock solutions (room temperature) of separating buffer (1.5M Tris-HCl, pH 8.8), stacking buffer (1M Tris-HCl, pH 6.8), SDS 10%, acrylamide/bis (see Note 3) solution (40%, 37.5:1 with 2.6% C) and ammonium persulphate 10% (stored at 4°C for 15 days max). N,N,N,N¢-Tetramethylethylenediamine (TEMED) is used pure. All products come from Bio-Rad. 2. For each electrophoresis, prepare 0.1 L of 10X running buffer (125 mM Tris buffered with 960 mM glycine, 0.5% w/v SDS), which can be stored at room temperature. 3. Laemmli-type loading buffer “2XU”: (2x) 100 mM Tris-HCl, pH 8.0, 8M urea, 4% SDS, 1.4M β-mercaptoethanol, 0.0025% bromophenol blue. The solution is stored at −20°C and aliquoted to freeze/thaw ten times maximum. 4. Prestained molecular weight markers: Kaleidoscope markers (Bio-Rad).

2.8. Western Blotting

1. Transfer buffer: 10 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid), pH 11.1, 10% methanol (see Note 4). Prepare fresh and use cold, with a cooling ice bag during transfer. 2. Polyvinyl fluoride membrane (PVDF (Polyvinylidene fluoride), Millipore) and 3 MM chromatography paper (Whatman) or nitrocellulose membrane. 3. Tris-buffered saline with Tween (TBS-T): Prepare a 10X stock with 1.37M NaCl, 27 mM KCl, 250 mM Tris-HCl, pH 7.4, 1% Tween-20. Dilute with water for use. 4. Primary antibody dilution buffer: TBS-T supplemented with 2% (w/v) fraction V bovine serum albumin (BSA). Primary antibody BXP21 (Millipore/Chemicon) is used 1/250 diluted. 5. Secondary antibody antimouse immunoglobulin G (IgG) rabbit conjugated to horseradish peroxidase (DakoCytomation) is used 1/3,000 diluted. 6. Enhanced chemiluminescent (ECL) reagents (GE healthcare) and Bio-Max ML film (Kodak) are used for revelation.

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3. Methods 3.1. Molecular Biology

Cloning of Drosophila NinaA and human BCRP into the pFastBac Dual plasmid (see restriction map in Fig. 7.1, panel A) is achieved using classical methods of molecular biology described in the booklet included in the Bac to Bac kit and in (17). NinaA was cloned between the BamH I and EcoR I restriction sites inside the polylinker region controlled by the PPH (Polyhedrin promoter) promoter from a DNA fragment produced by PCR using the wild-type gene as template, amplified with the 5¢ and 3¢ primers

kbp 6 4 3 2.5 5

SV40 pA pFastBac Dual

Tn7L

Ampicillin/ Gentamicin resistance

Tretacycline resistance

M 1

– – – – –

2 3 4 5341 4627 3274

2560

Kanamycine resistance

PCR

helper

helper

lacZ

HSV tk

bacmid

bacmid

pfastbacdual

recombinant

E.coli (Lac Z+)

Tn7R

Cloning

B

A

E.coli (Lac Z-)

C

Bacmid extraction

Virus amplification, Titration, Protein production

F

E

D

Fig. 7.1. Scheme of protein expression based on the Bac to Bac™ system. (a) The Drosophila protein chaperone NinaA (714 bp) and human BCRP (2,067 bp, including additional codons at the 5¢ end to insert affinity and detection tags) — or the membrane protein of interest — are cloned into the pFastBac™ Dual shuttle plasmid, into each polylinker region under the control of the Pp10 and PPH promoters, respectively. Dotted region corresponds to the fragment DNA inserted into the bacmid by recombination, between Tn7R and Tn7L regions, indicated in the scheme by a cross. (b) Competent DH10Bac™ Escherichia coli bacteria are then transformed with the plasmid, and positive transformants are selected by ampicillin/gentamicin resistance, in addition to tetracycline and kanamycin resistance, which allows maintaining the bacmid and helper plasmid, respectively, already present in this strain. (c) Transposition occurs by the transposase encoded in the helper plasmid. Bacmid having incorporated the foreign genes are screened by PCR using primers flanking the region of transposition: lane M molecular weight marker, lane 1 unmodified bacmid giving a PCR product of 2,560 bp, lane 2 bacmid including human BCRP (4,627 bp), lane 3 bacmid including NinaA (3,724 bp), lane 4 bacmid integrating both human BCRP and NinaA (5,341 bp). (d) Bacmid of each construct is prepared using a method dedicated to large DNAs (see Section 7.3.). (e, f ) Sf 9 insect cells are then transfected and checked for expression and virus titration as described in Section 7.3. (Scheme adapted from the Bac to Bac™ booklet. With permission).

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5¢-ACCGGATCCCCGCAAAATCATGAAGTCATTGCTCAATCGGATAAT and 5¢-AATGGAATTCAGCAGTACATGTTGAGCT (coding sequence is underlined, stop is double underlined; bold characters indicate restriction sites). Human BCRP was subcloned from a pTriEx construct in which the gene is already under the control of the Pp10 promoter, using the Pac I (5¢ end, cutting inside the Pp10 region) and Xho I (3¢ end) restriction sites. The following plasmids including the different constructs were generated: (1) pFastBac Dual (original plasmid), (2) pFastBac Dual–BCRP, (3) pFastBac Dual–NinaA, (4) pFastBac Dual–NinaA + BCRP. Once constructed, each plasmid was checked by sequencing and used to transform competent DH10BAC E. coli bacteria (Fig. 7.1, panel B). Positive clones, in which transposition between pFastBac Dual and the Bacmid has occurred, are selected by the white/ blue screen, with transposition leading to an insertion inside the gene coding for the β-galactosidase. Then, they are screened by PCR as follows: 1. Pick clones resistant to antibiotics (ampicillin, kanamycin and tetracycline) on a new LB (plus antibiotics) plate with a toothpick wetted into 10 mL water into a PCR tube. 2. Boil the solution for 10 min and withdraw 1 mL used as template for the PCR. 3. Carry out the PCR with M13 forward (−40) and M13 reverse primers (included in the Bac to Bac kit), located 128 bp upstream and 145 bp downstream from the locus of insertion, respectively. 4. Each construct is characterized by a given size PCR product as shown in Fig. 7.1, panel C: (1) unmodified bacmid giving a 2,560-bp PCR product, (2) bacmid including human BCRP (4,627 bp), (3) bacmid including NinaA (3,724 bp), and (4) bacmid integrating both human BCRP and NinaA (5,341 bp). 5. Extract each bacmid from positive clones using the kit for large DNA preparation PureLink HiPure Plasmid Midiprep (Fig. 7.1, panel D) and used to transfect Sf9 insect cells. 3.2. Sf9 Cell Transfection with Bacmids

1. In a T-25 flask (25 cm2, about 7.106 cells at confluence), dilute 500 mL of (liquid N2 frozen) stock cell suspension into 5 mL of Sf-900 II SFM medium supplemented with 10% FBS and 1% penicillin and streptomycin (both optional but recommended) and incubate for 2 days at 27°C. 2. Cells are then suspended in the liquid medium by tapping the flask, and 2.5 mL are withdrawn and mixed with 7.5 mL of fresh medium and incubated for 2 additional days in the same conditions.

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3. This initial culture is amplified in T-75 (75 cm2, about 15 ×฀106 cells at confluence) or a T-225 (225 cm2, about 50 ×฀106 cells at confluence) flask, seeded at 10 and 30 ×฀106 cells, to obtain enough cells for achieving transfection. Cells are counted with a microscope. 4. Spread 106 fresh cells in each well of a six-well culture plate (9 ×฀6 cm2/well, about 2.5 ×฀106 cells at confluence), one well for each assay of transfection plus a negative control (C−) in which no bacmid will be added. 5. Incubate the cells 1 h at 27°C to allow adhesion. 6. During this time, prepare for each trial of transfection the following mix: a. In a sterile Eppendorf tube A, add 1 mg recombinant bacmid/100 mL unsupplemented 1X Grace’s insect medium. b. In a sterile Eppendorf tube B, add 6 mL of cellfectin/100 mL unsupplemented 1X Grace’s insect medium. c. Mix each solution gently (three times by inversion) and pour the contents of tube B into tube A. Mix gently and incubate for 30 min at room temperature. 7. At the end of 1 h incubation of the cells, withdraw the medium from each of the wells and wash two times with 2 mL of unsupplemented 1X Grace’s insect medium. 8. Add 800 mL of unsupplemented 1X Grace’s insect medium to each DNA/lipofectin mix; the total volume will be 1 mL. Mix gently and add the mix gently to the corresponding well; the cells are then incubated at 27°C for 5 h, a time necessary to allow penetration of the bacmid. 9. The mix is then discarded and replaced by 2 mL of Sf-900 II SFM medium supplemented with 10% FBS and 1% penicillin and streptomycin, followed by a 72 h-incubation at 27°C. 10. The virus population generated from each assay is withdrawn as follows: In each well, cells are suspended by gentle pipetting and then pelleted by centrifugation at 500g for 10 min at room temperature. The 2 mL supernatant containing the baculovirus is transferred to a new sterile tube, constituting the generation called P1; it is stored at 4°C in the dark. 11. The cell pellet harvested at this stage from each 9.6-cm2 well of should give at 72 h postinfection about 2 ×฀5.106 cells at confluence. This is enough to check the expression of the protein of interest by SDS-PAGE and Western blot since about ten times less (2 ×฀105 cells, about 10 mg protein) are loaded onto the gels (see section 2.7 and 2.8). Protein expression is illustrated in Fig. 7.2a and detailed below.

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a

b

c

Fig. 7.2. Expression of BCRP in insect cells as a function of time, molecular chaperone and type of cell strain. Endpoint dilution. (a) Expression of BCRP in insect Sf 9 cells is carried out as described in Section 7.3.4. Cells were infected with a bacmid from the P3 population bearing pFastBacDual, alone (C−), with the gene coding for BCRP (BCRP) or with the genes coding for BCRP and NinaA (BCRP + NinaA) and grown for 24, 48 and 72 h postinfection in a six-well plate. BCRP expression was then analysed by SDS-PAGE and Western blot loading 10 mg of proteins onto a 10% SDS-PAGE (see Section 7.3.5.). BCRP is revealed with the antibody BXP21 diluted to 1/250, and the film was exposed for 5 min before revelation. The arrow indicates the position of migration of BCRP. (b) Compared expression of BCRP in High Five and Sf 9 insect cells. The expression is carried out as in A for 72 h and revealed as above. (c) Endpoint dilution. Dilution of the P3 stock suspension is carried out as indicated on the panel a and used to infect Sf 9 insect cells. Expression is checked after 72 h incubation as above.

3.3. Virus Amplification

The initial stock P1 is used to amplify the virus. Roughly, one can estimate that this solution corresponds to 5.106 plaque-forming units (PFU)/mL, with this value increasing tenfold at each generation. To generate the P2 stock solution (e.g., 30 mL using a T-225 flask seeded with 30 ×฀106 cells), the volume of P1 to add can be calculated using Equation 7.1: Volume of virus solution (mL) = (MOI × Number of cells)/ Solution virus titre, PFU/mL (Equation 7.1) where MOI is the multiplicity of infection estimated to 0.1. ⇔ 0.1 × 30.106 (cells)/5.106 (PFU/mL) = 0.6 mL P1 The culture is carried out as for P1 and harvested 72 h after infection. For large cultures, it is necessary to generate a P3 stock, infecting 5 T-225 flasks seeded at 30 ×฀106 cells with 60 mL of the P2 solution (thus ten times less than for generating P2 from P1), harvesting about 150 mL 72 h after infection. Each stock is stored at 4°C in the dark.

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3.4. Analysis of Protein Expression by SDS-PAGE and Western Blot

At each step, P1 to P3, the expression of the protein is checked by immunoblot after migration on SDS-PAGE and transfer on PVDF membrane. 1. Generate the separating gel (3.4 mL) of a 10% SDS-PAGE by mixing 1.92 mL of water, 1 mL of 40% acrylamide bisacrylamide solution, 1 mL of 1.5M Tris-HCl pH 8.8, 40 mL of 10% SDS, 40 mL 10% ammonium persulphate and 1.6 mL TEMED. Pour the Bio-Rad Mini-Protean 3 device to be 8 mm under the bottom of the wells. Add 200 mL of water at the surface of the gel for preventing the formation of waves. Do not use organic solvent for this step as membrane proteins have a tendency to interact with it. Polymerization occurs in 30 min at room temperature (22°C). 2. Generate the 5% stacking gel by mixing 1.46 mL of water, 0.25 mL of 40% acrylamide bisacrylamide solution, 0.25 mL of 1.5M Tris-HCl pH 6.8, 20 mL of 10% SDS, 20 mL 10% ammonium persulphate and 2 mL TEMED. 3. During polymerization of the stacking gel, prepare the protein samples as follows: a. Usually, 10 mg protein (estimated by the bicinchoninic acid method [18]) are loaded onto the gel, corresponding to a protein content of 2 ×฀105 cells. b. Pellet 4.105 cells and suspend them into 10 mL water and 10 mL 2XU Laemmli-type buffer. Allow the cells to be lysed and membrane proteins fully solubilised by 1-h incubation at room temperature. Do not heat the proteins, which tends to aggregate them, especially glycosylated ones. 4. Load 10 mg protein/10 mL onto the stacking gel and run for about 1.5 h at 25 mA and 120 V at room temperature. 5. During migration, a. Prepare the CAPS buffer for transfer and cool it at 4°C. b. Cut a 5 × 8 cm piece of PVDF membrane and six pieces of the same dimension of 3 MM chromatography paper. 6. After electrophoresis, transfer the protein from the SDS-PAGE to the PVDF membrane as follows: a. Incubate the gel in 5 mL of cold transfer buffer for 5 min. b. Wet the PVDF membrane with methanol briefly and then for 5 min in the cold transfer buffer. c. Prepare the transfer sandwich, built by superposing successively three paper sheets briefly wet in the transfer buffer, the acrylamide gel, the PVDF membrane (load the PVDF membrane onto the gel only one time since some proteins transfer quickly), and again three wet paper sheets.

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d. Add the ice cube to the Mini-Protean 3 transfer device and a magnetic fly and transfer for 1 h at 250 mA and 100 V under agitation to optimize cooling. e. After transfer, block the membrane for 1 h into 20 mL of TBS-T buffer containing 2% BSA. f. Add the primary antibody to the solution and incubate for an additional 1 h. g. Wash three times with 20 mL of TBS-T buffer. h. Incubate with 20 mL of TBS-T buffer containing 2% BSA and the second antibody for 1 h. i. Wash three times with 20 mL of TBS-T buffer. j. Withdraw the buffer and incubate with a 1:1 mix of 2 mL ECL solutions A and B for 5 min and expose onto a sensitive Kodak film for 1–10 min depending on the antibodies. A typical result is illustrated in Fig. 7.2. 3.5. Optimization of BCRP Expression in Insect Cells

Once the stock of P3 is obtained, it is useful to optimize the time of infection, check the effect of the molecular chaperon NinaA, check other insect cells and set up the dilution level of the virus stock for further expressions, here done by the method of endpoint dilution.

3.5.1. Optimization of the Time of Expression

1. In a six-well plate, seed in triplicate 2 mL of Sf-900 II SFM medium supplemented with 10% of FBS and 1% penicillin and streptomycin with 1.106 Sf9 cells and incubate for 2 h at 27°C before infection with 2 mL (1/100 dilution) with the P3 (pFastBac Dual alone or with BCRP or BCRP and NinaA), followed by a further incubation of 24, 48 and 72 h postinfection. 2. At the given time, cells are pelleted, solubilised and checked for BCRP expression by SDS-PAGE and Western Blot as described. The result is illustrated in Fig. 7.2a. It shows that the expression increases with time. Note that longer times of expression can be tested, such as 96 h.

3.5.2. Effect of Chaperon NinaA on BCRP Expression

As observed in Fig. 7.2a, NinaA plays the role of molecular chaperone for BCRP, influencing the level of expression and the homogeneity of BCRP. In absence of the chaperone, BCRP appears as two bands on the gel (lane 72 h), the upper being the most abundant, while when NinaA is co-expressed, BCRP appears as only one band corresponding to the upper one. It has been established that the lower band of BCRP produced in insect cells corresponds to an inactive form that comes from neither proteolysis nor differential glycosylation (19). The effect of NinaA on BCRP expression is thus positive since it tends to favour the folding

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of the protein. NinaA has a prolyl cis-trans isomerase activity, which could explain the observed effect, considering that BCRP has two proline residues located in the third span of the membrane domain for which a cis-trans isomerisation could have an impact on the folding of the protein. Note that in addition the co-expression of NinaA with BCRP leads to a two- to threefold increase of expression of BCRP. 3.5.3. Expression of BCRP in Sf 9 and Hi5 Insect Cells

One can use different strains for checking expression of the target protein. Initial transfection is carried out with Sf9 insect cells, but Hi5 cells are described to get a higher level of protein expression. Figure 7.2b illustrates the comparison of BCRP expression in the presence of NinaA in both types of cells. As expected and shown, Hi5 cells produce a higher amount of BCRP than Sf9 cells. However, most of the protein produced corresponds to the lower band species, although the co-expression of NinaA. The level of the upper band of BCRP is the same in both types of cells, indicating that Sf 9-type cells used with NinaA constitute the best choice for expressing BCRP.

3.5.4. Endpoint Dilution

Once the P3 solution is generated, check the dilution at which the stock of virus will be used. 1. Starting with P3, dilute the viral suspension (Bacmid pFastBac Dual - BCRP + NinaA) to 4, 10, 50, 102, 104, 106 and 108 times into 2 mL Sf-900 II SFM medium supplemented with 10% of FBS and 1% penicillin and streptomycin and pour a six-well plate loaded with 1.106 Sf 9 cells. Incubate for 72 h at 27°C. 2. At the end of the culture, withdraw 4.105 cells and check BCRP expression by SDS-PAGE and Western Blot as described. The result is illustrated in Fig. 7.2c, which shows that a 1/100 dilution is enough to maintain a high level of expression.

3.5.5. Scale-up in Spinners

Liquid cultures are carried out in 1-L spinners seeded with 5.109 Sf9 cells infected with 10 mL of P3 stock (10−2 dilution) and incubated for 72 h at 27°C and 60 rpm on a H + P Biomag Biomodule 40B. This leads routinely to 2 mg BCRP/L.

4. Notes 1. Baculovirus is not infectious for humans; its culture does not require class II-type equipment. However, depending on the gene expressed, it can be necessary to use this type of classified room. 2. Ampicilline should be prepared fresh to a maximal efficiency.

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3. Acrylamide is neurotoxic when unpolymerised, thus handle with gloves. 4. Methanol is neurotoxic. References 1. Jarvis DL, Finn EE (1996) Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat Biotechnol 14:1288–92 2. van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9:112–124 3. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotech 23:567–575 4. Blissard GW, Rohrmann GF (1990) Baculovirus diversity and molecular biology. Annu Rev Entomol 35:127–155 5. Smith GE, Vlak JM, Summers MD (1982) In vitro translation of Autographa californica nuclear polyhedrosis virus early and late mRNAs. J Virol 44:199–208 6. Jarvis DL, Kawar ZS, Hollister JR (1998) Engineering N-glycosylation pathways in the baculovirus-insect cell system. Curr Opin Biotechnol 9:528–533 7. Marheineke K, Grunewald S, Christie W, Reilander H (1998) Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection. FEBS Lett 441:49–52 8. Gilbert RS, Nagano Y, Yokota T, Hwan SF, Fletcher T, Lydersen K (1996) Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium. Cytotechnology 22:211–216 9. Dean M, Hamon Y, Chimini G (2001) The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 42:1007–1017 10. Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage H, Dietel M, Greenberger L, Cole SP, Doyle LA (1999) Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 91:429–433 11. Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, Bates

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SE (2000) The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 113(Pt 11):2011–2021 Ozvegy C, Varadi A, Sarkadi B (2002) Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation. J Biol Chem 277:47980–47990 McDevitt CA, Collins RF, Conway M, Modok S, Storm J, Kerr ID, Ford RC, Callaghan R (2006) Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2. Structure 14:1623–1632 Pozza A, Perez-Victoria JM, Sardo A, AhmedBelkacem A, Di Pietro A (2006) Direct interaction with purified breast cancer resistance protein ABCG2 indicates arginine-482 involvement in drug transport, not in binding. Cell Mol life Sci 63:1912–1922 Baker EK, Colley NJ, Zuker CS (1994) The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J 13:4886–4895 Lenhard T, Reilander H (1997) Engineering the folding pathway of insect cells: generation of a stably transformed insect cell line showing improved folding of a recombinant membrane protein. Biochem Biophys Res Commun 238:823–830 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85 Pozza A, Perez-Victoria JM, Di Pietro A (2009) Overexpession of homogeneous and active ABCG2 in insect cells. Protein Expr Purif 63(2):75–83

Chapter 8 Expression of Membrane Proteins in Drosophila Melanogaster S2 Cells: Production and Analysis of a EGFP-Fused G Protein-Coupled Receptor as a Model Karl Brillet, Carlos A. Pereira, and Renaud Wagner Abstract In the process of selecting an appropriate host for the heterologous expression of functional eukaryotic membrane proteins, Drosophila S2 cells, although not yet fully explored, appear as a valuable alternative to mammalian cell lines or other virus-infected insect cell systems. This nonlytic, plasmid-based system actually combines several major physiological and bioprocess advantages that make it a highly potential and scalable cellular tool for the production of membrane proteins in a variety of applications, including functional characterization, pharmacological profiling, molecular simulations, structural analyses, or generation of vaccines. We present here a series of protocols and hints that would serve the successful expression of membrane proteins in S2 cells, using an enhanced green fluorescent protein (EGFP)/G protein-coupled receptor (EGFP-GPCR) as a model. Key words: Drosophila S2 cells, heterologous expression, membrane proteins, recombinant GPCR

1. Introduction “A relatively recently developed cell technology for heterologous gene expression has been developed with Schneider 2 (S2) Drosophila cells. Although still lacking further detailed studies, this system offers, as a premise, some interesting advantageous possibilities such as, the high cell density easily attained (50–100 times higher when compared to mammalian cells), the growth of cells in suspension (circumventing the use of microcarriers), the use of inducible promoters (concentrating the protein production to a period of time and avoiding degradation) and the establishment of a continuous bioprocess (in contrast to bioprocess involving cell lysis induced by viral vectors).” These introductory statements were extracted from the foreword for a special issue of

I. Mus-Veteau (ed.), Heterologous Expression of Membrane Proteins, Methods in Molecular Biology, vol. 601 DOI 10.1007/978-1-60761-344-2_8, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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Cytotechnology on heterologous gene expression in Drosophila melanogaster cells (1) published in May 2008; it brings a representative illustration of how this system is increasingly used for the synthesis of a great variety of proteins. In particular, S2 cells are regularly proving to be an efficient and adequate system for the expression of demanding candidates. These cells represent a highly versatile expression system useful for both the functional analysis of genes that are naturally expressed at low levels and the production of significant amounts of recombinant proteins that are then amenable to biochemical or structural studies. This typically meets the requirements needed for the expression of functional membrane proteins, as exemplified in several studies focusing not only on monotopic membrane proteins such as lectins (2), enzymes (3), or viral membrane glycoproteins (4–6), but also on more complex candidates, including transporters (7, 8), ion channels (9–14), G protein-coupled receptors (GPCRs) (15–28), or even membrane protein complexes (13, 29). From all these studies conducted for functional characterization, pharmacological profiling, molecular simulations, structural analyses, or vaccinology purposes, the S2 cells constantly appeared as an efficient and valuable expression system due to a number of appealing properties. 1.1. The Drosophila S2 Cell Line

The S2 cells were isolated more than 30 years ago from primary cultures of Drosophila melanogaster embryos (30) as immortalized nontumorgenic cells, which may be derived from blood cells as recently suggested (31). S2 cells usually grow in a loose monolayer manner with a slight tendency to pile up, and they present epithelial-like characteristics. In contrast to mammalian cells, S2 cells are advantageously grown at room temperature using simple media and do not require any CO2 supplementation. One specific characteristic of S2 cells over other cell lines is their ability to grow to high cell densities, routinely reaching 3 × 107 cells/mL, which is about tenfold higher than what can be attained with other insect cells. Using slightly modified media, S2 cells can also be grown in suspension and can conveniently accommodate largescale culturing using bioreactors (16, 32). As a higher eukaryotic host, S2 cells possess all the mammalianlike cell machineries for gene expression, protein processing, and trafficking, including finicky posttranslational modifications that may be critical for proper maturation, localization, and function of demanding proteins, even if N-glycosylations are not strictly similar (2). In addition, S2 cells have been shown to lack endogenous surface proteins such as voltage-gated channels (33), which makes them particularly appropriate for the expression and functional studies of ectopic channels or neurotransmitter receptors.

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Expression of recombinant proteins in S2 cells relies on a nonlytic plasmid-based system that thus provides additional benefits over virus-infected insect systems. S2 cells can actually be transiently or stably transfected using a series of plasmids bearing constitutive or inducible promoters, offering a significant panel of flexible expression conditions to be tested. The most widely used vectors contain either the constitutive promoter from the actine 5C gene (pAc5) (34) or the copper-inducible metallothionein promoter (pMT) (35), the last by far preferred for membrane protein expression. Stable cell lines are usually obtained after 3–4 weeks by cotransfecting the expression vector together with a selection vector bearing a drug-resistance marker, with the most common one the hygromycin B-phosphotransferase gene allowing for hygromycin B selection. A remarkable characteristic of S2 stable cell lines is the presence of multicopy insertions of the two vectors (several hundreds of copies) (36), a substantial gene amplification that results in an increased expression of the protein of interest. While this system is now well established and commercially available (Drosophila Expression System, DES®, from Invitrogen), several alternative expression strategies utilizing S2 cells have been explored, some of them successfully expressing proteins in nonlytic, single or multiple, baculovirus infection procedures (37, 38), some other achieving stable S2 cell lines with a single vector (39, 40). In this chapter, we propose to use a human mu opioid receptor (hMOR) N-terminally fused to the enhanced green fluorescent protein (EGFP) as a model membrane protein to illustrate a series of protocols that allow handling of a regular S2 cell expression system. In addition to the description of generic procedures for S2 cell culturing, this EGFP-fused expressed GPCR offers a good opportunity to exemplify some membrane protein-specific experiments that can be performed for assessing both the protein yield and functionality.

2. Materials 2.1. Cell Culture Procedures

1. The Drosophila melanogaster S2 cells and the pCoHYGRO and pMT/BiP vectors were obtained from Invitrogen.

2.1.1. S2 Cell Line and Vectors

2. The coding sequence of the mu opioid receptor was cloned into a pcDNA3.1 vector; the sequence of the EGFP was from a pEGFP-C3 vector (Clontech). 3. Use common materials and enzymes for standard cloning methods.

2.1.2. Culturing and Freezing S2 Cells

1. Laminar flow bench, incubator. 2. Hemacytometer and trypan blue.

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3. InsectXpress complete medium: InsectXpress (Cambrex), 10% fetal bovine serum (FBS) heat inactivated 30 min at 65°C, 0.2% Pluronic F-68, 50 mg/mL gentamicin. 4. Dimethyl sulfoxide (DMSO). 5. Sterile 25-, 75-, and 175-cm2 flasks, six-well plates, and pipets. 6. Sterile 15-mL conical tubes and cryovials. 2.1.3. Suspension and Bioreactor Cell Culturing

1. Sterile T75-cm2 flasks and pipets. 2. InsectXpress complete medium. 3. Heater/shaker incubator. 4. Hemacytometer and trypan blue. 5. Sterile 500-mL shake flasks (Schott-type bottles fit well). 6. Sterile 1-L Schott-type bottle with double tubing through the septum of an open-top cap, one equipped with a 0.22-mm filter and one that can be connected directly to an inlet pipe of the reactor. 7. We use a 3-L reactor from Applikon® with a working volume of 2.7 L; other suitable reactors can be used. The reactor bears three marine impellers and is sparging aerated (1.8 L/min) through filters with a pore size of 0.22 mm. DO, pH, stirrer speed, and temperature parameters are controlled and recorded using dedicated software (Bioxpert, Applikon).

2.2. Procedures for Membrane Protein Expression and Analysis

1. Laminar flow bench, incubator.

2.2.1. Cell Transfection, Clone Selection, and Induction of Expression

5. Hygromycin.

2.2.2. Cell Sorting

1. Laminar flow bench, incubator.

2. Sterile six-well plate, combitips, and pipets. 3. Sterile 150 mM NaCl. 4. JetPEI™ DNA transfection reagent (Polyplus Transfection). 6. CuSO4.

2. Sterile and degassed PEB buffer: PBS supplemented with 5 mM ethylenediaminetetraacetic acid (EDTA) and 1% bovine serum albumin (BSA). 3. Anti-EGFP monoclonal antibody (see Note 1). 4. Goat antimouse immunoglobulin G (IgG) magnetic beads from Miltenyi Biotec (Bergisch Gladbach, Germany). 5. MACS® column from Miltenyi Biotec with the magnet support. 6. Sterile 12-well plates.

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1. Lysis buffer: 50 mM Tris-HCl, pH 8, 1 mM EDTA. 2. Potter homogenizer. 3. Benchtop ultracentrifuge. 4. Membrane buffer: 50 mM Tris-HCl, pH 7.4, 320 mM sucrose. 5. 26-gauge needle. 6. BCA Protein Assay kit (Pierce).

2.2.4. Western Blot Analysis

1. Equipment for polyacrylamide gel electrophoresis (PAGE) and blotting device (from Bio-Rad or another supplier). 2. 10% sodium dodecyl sulfate (SDS) polyacrylamide gel. 3. Molecular weight marker. 4. MPBST buffer: 3% nonfat dried milk in PBS supplemented with 0.5% Tween-20. 5. 1/1,000 anti-EGFP monoclonal (Boehringer Ingelheim) in MPBST. 6. 1/2,000 antimouse IgG horseradish peroxidase (HRP)linked whole antibody from sheep (GE Healthcare) in MPBST. 7. Supersignal West Pico chemiluminescent substrate (Pierce).

2.2.5. Fluorescence Measurements

1. Insect Elliot buffer: 130 mM NaCl, 5.5 mM KCl, 1.2 mM MgCl2, 4.2 mM NaHCO3, 7.3 mM NaH2PO4, and 20 mM HEPES, pH 6.2. 2. Hemacytometer and trypan blue. 3. A 1-mL cuvette with magnetic stirring (Hellma). 4. Spectrofluorimeter (PTI).

2.2.6. Ligand-Binding Assays

1. PBS buffer supplemented with 320 mM sucrose. 2. 50 mM Tris-HCl, pH 7.4, 320 mM sucrose. 3. Polyethylenimine. 4. Nunc® minisorp tubes. 5. [3H]-Diprenorphine (PerkinElmer). 6. Naloxone (Sigma Aldrich). 7. GF/B filters. 8. Brandel® cell harvester (Alpha Biotech). 9. Ultima Gold scintillation cocktail (PerkinElmer). 10. Hinge-cap vials. 11. Tricarb® scintillation counter (PerkinElmer).

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3. Methods 3.1. Cell Culture Procedures

This section describes the different methods required for culturing S2 cells, from T-flask culturing (initiating, maintaining, and freezing cells) to suspension culturing in shake flasks or in a bioreactor.

3.1.1. Culturing S2 Cells

This method describes how to start and maintain the S2 culture from 1 mL of liquid nitrogen-frozen stock containing 1 × 107 cells (see Note 2). 1. Remove the vial of cells from liquid nitrogen and thaw quickly in your hand. 2. Decontaminate the outside of the vial with 70% ethanol and transfer quickly in 5 mL of room temperature InsectXpress complete medium. 3. Centrifuge the cells at 100g for 2–3 min and remove the medium. Resuspend the cells with 5 mL of InsectXpress complete medium and plate them in a T25-cm2 flask. 4. Incubate at 27°C under normal atmosphere for 24 h. 5. The next day, remove the medium from the flask and carefully add 5 mL of InsectXpress complete medium. Incubate at 27°C until the cells reach up to 20 × 106 cells/mL. This may take 3 or 4 days. 6. To maintain the culture, S2 cells are usually split twice a week in InsectXpress complete medium at a 1:5 to 1:10 dilution into new culture vessels. Cells can be split at a final density of 2 × 106 cells/mL. Never start a new culture at a density below 0.5 × 106 cells/mL.

3.1.2. Freezing S2 Cells

1. Remove the cells from the flask; tap several times to remove the cells attached to the surface. Briefly pipet the cell suspension up and down to break up clumps of cells. 2. Count a sample of the cell suspension in a hemacytometer using a standard trypan blue exclusion assay to determine the number of cells per milliliter and the viability (95–99%). 3. Pellet the cells by centrifuging at 100g for 2–3 min in a tabletop centrifuge at 4°C. Remove the supernatant from the cell pellet. 4. Resuspend in 90% FBS and 10% DMSO at a density of 1 × 107 cells/mL. 5. Aliquot 1 mL of the cell suspension per cryovial. Place the vials in a polystyrene box and allow the cells to freeze in a −80°C freezer for 24 h before transferring the vials to liquid nitrogen for long-term storage.

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1. Prepare a T75-cm2 flask S2 cells. When the culture reaches confluence, almost 20 × 106 cells/mL, inoculate a 500-mL bottle with 10 mL of the cell suspension and add 90 mL of InsectXpress complete medium (see Note 3). 2. Incubate the bottle in a heater/shaker incubator at 110 rpm and 27°C. 3. After 72 h, count a sample of the cell suspension in a hemacytometer with trypan blue to determine the number of cells per milliliter and the viability. The cell suspension is supposed to reach a density of almost 30 × 106 cells/mL with 95–99% viability. 4. The suspension culture can be then amplified and tested for production in larger volumes.

3.1.4. Bioreactor Cell Culture

1. Autoclave the empty Applikon bioreactor vessel, as well as a 1-L bottle equipped with the needed tubing (filtered vent hole and connection to the bioreactor). 2. Prepare 100 mL of a S2 cell suspension culture in InsectXpress complete medium as described. 3. Count a sample of the cell suspension in a hemacytometer with trypan blue to determine the number of cells per milliliter and the viability. 4. In the bottle prepared at step 1, dilute the cell in fresh InsectXpress complete medium to end with 1 L at a final concentration of 2.5 × 106 cells/mL and sterilely connect the bottle to the bioreactor. 5. Add the fresh cell suspension to the bioreactor by pressuring the bottle connected to the bioreactor (see Note 4). 6. Start the bioreactor culture using the following settings: 20% dissolved oxygen (DO) (see Note 5), 120 rpm stirrer speed, and a temperature controlled at 27°C. The DO can be controlled by sparging air or oxygen. 7. During the whole run, samples can be sterilely collected via a pipe in the culture fluid and transferred to sample vessels by pressuring the bioreactor for analysis (see Note 6).

3.2. Membrane Protein Expression

In this section, we describe how to transfect S2 cells and generate stable recombinant cell lines for the heterologous expression of membrane proteins.

3.2.1. Expression Vector

Due to space limitation reasons, we will not go into detail for the construction of the expression vector. Briefly, the BiP signal sequence, the EGFP, and hMOR open reading frames (ORFs) were cloned in frame under the control of the pMT promoter in the pMT/BiP vector using standard cloning procedures

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(see Note 7). The resulting construct was sequence checked and, as was done for the pCoHYGRO vector, was Escherichia coli amplified and purified using a standard DNA miniprep kit. 3.2.2. Transfection for the Generation of Stable Cell Lines

For S2 cell transfection, several reagents can be successfully used (lipofectin, lipofectamine, cellfectin, etc.) [41). We prefer to use the jetPEI reagent, which proved the most efficient in our hands (see Note 8). 1. One day before transfection, plate 1.5 × 106 S2 cells on a sixwell plate with InsectXpress complete medium (see Note 9). 2. Dilute 5–10 mg of the respective pMT construct and 0.25– 0.5 mg pCoHYGRO (1:20 ratio) into a final volume of 100 mL of 150 mM NaCl. Vortex gently and spin down briefly. 3. Dilute 10–20 mL of jetPEI solution into a final volume of 100 mL of 150 mM NaCl. Vortex gently and spin down briefly. 4. Add the 100 mL of jetPEI solution to the 100 mL of DNA solution all at once (important: do not mix the solution in the reverse order). 5. Vortex the solution immediately and incubate for 30 min at room temperature. 6. Remove the cell medium from the six-well plate and add 800 mL of fresh complete medium. 7. Gently add 200 mL of jetPEI/DNA mixture per well and incubate 24 h at 27°C.

3.2.3. Selection of Stable Cell Lines

1. At 24 h after transfection, remove the medium and start selection by adding hygromycin at a final concentration of 300 mg/mL. 2. Change medium containing hygromycin every 2 or 3 days. 3. Two weeks after starting the selection, progressively decrease the antibiotic concentration to 0. 4. Two weeks later, you can proceed to protein expression analysis after CuSO4 induction for each of your stable cell lines. Select the best clone and start the amplification.

3.2.4. Induction of Recombinant Protein Expression

Induction of expression is easily achieved by simply adding sterile copper sulfate to the medium to a final concentration of 700 mM, whatever the culture format used (T-flask, bottle or bioreactor). It is important to spend time in optimizing this step since major variations can occur, resulting in dramatic improvements of the yield and activity of the expressed membrane protein. Several parameters can be tested, such as time course postinduction (from 12 to 72 h, for instance), temperature of induction (i.e., 22–28°C), or concentration of CuSO4 (250 mM to 1 mM). In the particular

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case of GPCR expression, addition of chemical additives such as 0.5–4% DMSO or specific antagonist ligands during the induction phase often proves helpful (15) (see Note 10). 3.2.5. Cell Sorting

As frequently observed with the S2 system, stable cell lines obtained after clone selection are often considered polyclonal and not homogeneously expressing the desired protein. Separative techniques such as FACS experiments could then be helpful in enriching the cell lines in receptor-expressing subpopulations. We describe here a robust alternative immunogenic method that can be applied to most membrane proteins to be expressed and that does not require expensive equipment (see Note 11). 1. After CuSO4 induction of an S2 culture, pellet about 107 cells at 900 rpm for 3 min and resuspend the cells in 25 mL PEB buffer. 2. Incubate at 4°C for 10 min with PEB buffer supplemented with 1/200 anti-EGFP monoclonal antibody (see Note 1). 3. Wash twice with PEB buffer and resuspend the cells in 25 mL PEB buffer supplemented with 1/5 volume goat antimouse IgG magnetic microbeads. 4. Incubate at 4°C for 15 min. 5. Wash twice with PEB buffer, centrifuge the sample at 100g for 10 min, and resuspend in 500 mL PEB buffer. 6. Fix the MACS column onto its magnet, apply 500 mL of degassed PEB buffer on top of the column, and let the buffer run through. 7. Pipet the magnetically labeled cell suspension onto the column and collect effluent as the negative fraction. 8. Wash the column three times with 500 mL PEB buffer and collect total effluent as the negative fraction. 9. Remove column from its magnet and place the column on a new sterile collection tube. 10. Apply 1 mL of PEB buffer to the reservoir of the column and flush the cells using the plunger supplied. 11. Wash the sorted cells with InsectXpress complete medium and split in 12-well plates. 12. After amplification of the sorted cells, test protein expression by CuSO4 induction.

3.3. Analyzing Yield and Activity of the Expressed Membrane Protein

Several techniques can be employed for assessing the activity and the quantity of an expressed membrane protein, starting either from whole S2 cell material or from membrane preparation samples. The most common approaches to evaluate membrane protein expression include the enzyme-linked immunosorbent assay

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(ELISA) or ligand-binding assay for protein quantification, flow cytometry for evaluation of cells expressing the protein, confocal microscopy for analyzing the protein localization, spectrofluorimetry for the protein evaluation through fluorescence measurement, and Western blotting for initial qualitative analysis of the recombinant protein. At this step, the most helpful way, in terms of bioprocess optimization, is to express the amount of recombinant protein as evaluated by ELISA per number of cells in culture. After describing an adapted protocol for preparing total S2 membranes, we present in this section three methods suited to our EGFP–hMOR fusion that can be easily adapted to similar constructs. 3.3.1. S2 Cells Lysis and Membrane Preparation (See Note 12)

1. Centrifuge approximately 8 × 108 induced S2 cells at 100g for 3 min at 4°C. 2. Discard the supernatant and freeze the sample at −80°C. 3. Thaw the cells in 1 mL 50 mM Tris-HCl, pH 8, 1 mM EDTA. 4. Lyse the cells with 20 strokes in a Potter homogenizer. 5. Pellet the nuclei and the unbroken cells by centrifugation at 2,000g for 10 min at 4°C. Keep the supernatant. 6. Resuspend the pellet in 1 mL Tris-HCl, pH 8, 1 mM EDTA, lyse, and centrifuge again at 1,000g for 5 min at 4°C. 7. Pool both supernatants from steps 5 and 6 and ultracentrifuge at 100,000g for 30 min at 4°C. 8. Resuspend the membrane pellet in 500 mL of 50 mM TrisHCl, pH 7.4, 320 mM sucrose. 9. Homogenize through a 26-gauge needle and proceed to a protein concentration assay using the BCA kit. 10. Directly use samples for further analysis or store as aliquots at −80°C.

3.3.2. Western Blot Analysis

1. Mix the sample with Laemmli buffer for 30 min at room temperature before loading onto a 10% SDS-polyacrylamide gel and proceed to SDS-PAGE. 2. Electrotransfer the proteins onto a PVDF membrane at 60 V for 2 h. 3. Block the membrane in 3% nonfat dried milk, PBS, 0.5% Tween-20 (MPBST) for 30 min. 4. Incubate the membrane for 1 h in MPBST supplemented with 1/1,000 anti-EGFP or anti-hMOR monoclonal antibodies. 5. Wash the membrane three times for 5 min each in MPBST and incubate for 1 h in MPBST supplemented with 1/2,000 antimouse IgG HRP-linked whole antibody from sheep.

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6. Wash four times for 5 min each with MPBST and then detect the chemiluminescence using Supersignal West Pico chemiluminescent substrate according to the manufacturer’s instructions. 3.3.3. Fluorescence Measurements

As the protein is N-terminally fused to EGFP, fluorescence methods can be used directly on whole cells or subcellular fractions to visualize the expressed protein for different purposes (cytolocalization, quantification, etc.). We present here a method allowing the evaluation of the expression level in whole cells by directly recording the EGFP fluorescence. 1. In parallel cultures, induce recombinant and wild-type S2 cells. 2. Collect the cells from the flask and tap several times to remove the cells attached to the surface. Briefly pipet the cell suspension up and down to break up clumps of cells. 3. Count a sample of the cell suspension in a hemacytometer with trypan blue to determine the number of cells per milliliter and the viability. 4. Pellet the cells by centrifuging at 100g for 2–3 min in a tabletop centrifuge at 4°C. Remove the supernatant from the cell pellet. 5. Resuspend 107 cells in 1 mL of Insect Elliot Buffer. 6. Place the cell suspensions in a 1-mL cuvette with magnetic stirring and maintain at 21°C in the thermostated cell handler of a dedicated spectrofluorimeter, with slits sets to yield a bandwidth of 2 nm at excitation and emission. 7. After excitation at 470 nm, record the EGFP fluorescence from 490 to 750 nm. 8. Calculate the specific fluorescence of the sample by withdrawing the background of the wild-type cells to the fluorescence of the sample at 510 nm.

3.3.4. Ligand-Binding Assay

The typical GPCR activity ligand-binding assay can be carried out either on whole cells (present protocol) or on membrane preparations (see Note 13). In the latter case, 5–20 mg of membrane preparations can be used in each assay in place of whole cells. 1. After induction of recombinant S2 cells, prepare the cells as described in steps 2 to 4 in the previous protocol for fluorescence assay (see Section 3.3.3). 2. Wash the obtained cell pellet twice in PBS buffer supplemented with 320 mM sucrose. 3. Resuspend the cells in 50 mM Tris-HCl, pH 7.4, 320 mM sucrose at a final concentration of 5 × 106 cells/mL.

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4. Distribute 400 mL of the cell suspension in each immunosorp tube. 5. In tubes prepared for the determination of total binding activity, add 100 mL of 50 mM Tris-HCl, pH 7.4, 320 mM sucrose supplemented with a concentration range of [3H]-diprenorphine, typically from 0.05 to 3.2 nM final concentration (see Note 14). 6. In parallel tubes prepared for the determination of nonspecific binding, add the same solution as in step 8, supplemented with 2 mM Naloxone (see Note 14). 7. Vortex each tube and incubate for 40 min at room temperature. 8. Presoak GF/B filters with 0.1% polyethylenimine. 9. Filter the samples and wash twice with ice-cold 50 mM TrisHCl, pH 7.4, using a Brandel cell harvester. 10. Transfer the filters in hinge-cap vials and add 5 mL of Ultima gold scintillation cocktail. 11. Incubate 1 h in a dark place before counting the sample in a suitable scintillation counter. 12. Analyze the data to determine the specific ligand-binding values. This can be done using appropriate software such as GraphPad Prism.

4. Notes 1. Any other monoclonal antibody directed against the membrane protein of interest can work as well provided the detected epitope is exposed at the cell surface. 2. New stocks can be thawed every 4 months. 3. For an optimal suspension culture, we recommend using a working volume of 1:5 in shake flasks or bottles to provide suitable oxygen transfer through the medium surface to the cell culture during agitation (42). Also, whereas the Pluronic F-68 reagent is optional for classical culture in T-flasks, it is necessary to keep this reagent in suspension culture as it plays a crucial role for maintaining cell integrity. 4. If necessary, an antifoaming agent can be used, such as Contraspum 210 or Contraspum A 4,060 (Zschimmer and Schwarz) or any other antifoaming agents previously tested on an S2 cell culture.

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5. It is usually recommended to set the DO at 20%. We found, however, that lower DO (down to 5%) was also suitable and generated less foam. 6. Analyses of bioreactor samples typically include not only cell density and viability evaluations and determination of yields and activity in case of recombinant proteins but also enzymatic assays or nutrient consumption/metabolite production measurements to obtain information on the physiology of the cultured cells for bioprocess optimization. 7. For membrane proteins bearing an extracellular N-terminus, such as GPCRs, we recommend employing a BiP signal sequence fusion for better expression yields and protein activity. Alternatively, use expression vectors without the BiP sequence combined with a constitutive pAc promoter. Also, the selection gene (hygromycin) can be cloned into the expression vector, allowing a single transfection instead of a cotransfection as described here. 8. According to the manufacturer’s recommendations, use an optimal jetPEI ratio of 2 mL of jetPEI for 1 mg of plasmid DNA. 9. Contrary to other reagents, jetPEI is not affected by the presence of serum during transfection, so the InsectXpress complete medium can be used. 10. We recommend testing the different mentioned parameters that may affect the productivity and activity of the expressed protein separately and then combining in a second optimization round those exhibiting a positive effect (e.g., DMSO is believed to potentiate the effect of ligand supplementation). 11. A limiting dilution method for cell cloning can be laborious for S2 cells since they do not easily grow at densities lower than 103 cells/well, and a feeder cell methodology is not yet well developed for insect cells. 12. This protocol is designed for the preparation of total membranes. Further membrane fractionations may also be useful as they may allow the separation of active from nonactive membrane protein subpopulations, as illustrated in (14). 13. The present assay is designed for the mu opioid receptor but can be adapted for other receptors using appropriate ligands. Note also that the binding assay on whole cells will mainly detect receptors exposed at the cell surface, with the accessibility of intracellularly located receptors to ligand dependent on the hydrophobicity of the ligands used. 14. For each concentration of radioligand tested, we recommend carrying out the assay in triplicate.

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Chapter 9 Membrane Protein Expression in the Eyes of Transgenic Flies Valérie Panneels and Irmgard Sinning Abstract Eukaryotic membrane proteins are often difficult to obtain in sufficient amounts for structural studies using classical cell culture overexpression systems. This could be due to incomplete protein maturation and insufficient trafficking to the plasma membrane or to a cytotoxic effect of the recombinant membrane protein changing the overall metabolism. The method presented here takes advantage of the membrane stacks in the retina, where rhodopsin resides. The idea is to direct G protein-coupled receptors (GPCRs) and transporters to these membrane stacks and to express the target proteins in the retina of transgenic Drosophila melanogaster. Drosophila was chosen since fly genetics are well established and rather easily accessible. Metabotropic glutamate receptor mGluRa from Drosophila was among the first examples for GPCRs expressed in this system. It showed high expression yield, functionality and high homogeneity. When the same protein was expressed in Sf 9 cells, however, contamination by immature protein was a problem. Encouraged by this success, the fly system is now successfully used for a larger variety of membrane proteins, including mammalian GPCRs and transporters. Key words: Drosophila, EAAT2, fly genetics, glutamate transporter, GPCR, membrane protein, overexpression, photoreceptor cells, retina, rhodopsin, transporter

1. Introduction Many membrane proteins (MPs) are present in their native membrane only in minute amounts. Therefore, in most cases the amounts needed for biochemical and structural studies can only be obtained by recombinant expression using different established techniques (1–4). Rhodopsin is a remarkable exception as it is present in high amounts in the retina, where it is involved in vision. Besides this important function, it is the most abundant G proteincoupled receptor (GPCR) and has served as a prototype for structural and functional studies of this family. The structure of rhodopsin was the first X-ray structure of a GPCR (5) and could be I. Mus-Veteau (ed.), Heterologous Expression of Membrane Proteins, Methods in Molecular Biology, vol. 601 DOI 10.1007/978-1-60761-344-2_9, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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determined starting from membranes of the retina, where rhodopsin is densely packed (6, 7). How rhodopsin is targeted to the specialized membrane stacks in the photoreceptor cells — termed rod outer segment (ROS) in mammals or rhabdomeres in Drosophila — is not completely understood (8). Membrane insertion and targeting of rhodopsin must be efficient since rhodopsin — in addition to its abundance —shows a high turnover. Encouraged by these observations, we developed the expression of recombinant MPs in the eyes of transgenic animals as a new, alternative method for overexpression. Drosophila melanogaster was chosen as it offers the advantage of well-established fly genetics. Expression levels, kinetics and location of the target protein can be modulated and controlled using different promoters and a transactivating Gal4/ upstream activating sequence (UAS) system (9, 10). First, a stable fly strain containing a UAS for the ectopic yeast protein Gal4 followed by the target protein to be expressed is generated. Next, this fly is crossed with another strain named the driver strain and containing the “promoter-Gal4” transposon to generate a population expressing the gene under the control of Gal4. The glutamate receptor from D. melanogaster (mGluRa) was successfully expressed as the first GPCR using this method (11). It was shown to be functional, and its capacity to bind glutamate was shown to depend on cholesterol (12). Since this first study, the method was developed further using green fluorescent protein (GFP) fusion constructs that allow monitoring of expression, purification and localization of the protein. A larger number of GPCRs and transporters, not only from Drosophila but also mammals, were successfully expressed in the fly eyes, demonstrating the great potential of this method (described elsewhere). Here, we give a practical introduction to this new approach for the expression of MPs. Briefly, the MP constructs are cloned into the transposon cassette of a vector, which is injected into Drosophila embryos. Transformant flies obtained from random transposition into the genome are selected and stabilised by crossing with balancer flies. A driver fly for eye-specific induction of expression is crossed with the stable strain, and cultures are scaled-up. The heads of frozen flies are collected, and membranes are prepared for further functional or structural studies.

2. Materials 2.1. Membrane Protein Cloning

1. pUAST vector (gift from C. Desplan, New York, USA). 2. Human glutamate transporter EAAT2 gene construct (gift from M. T. Besson and S. Birman, Marseille, France). 3. Enhanced GFP (EGFP) gene construct (BioCat, Germany).

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4. Topo® TA Cloning® kit (Invitrogen). 5. Qiaprep® Spin Miniprep and QIAquick Gel Extraction kits (Qiagen). 6. T4-DNA ligase (Biolabs). 7. SOC medium: 0.5% Yeast Extract, 2% Tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose. 8. Luria-Bertani (LB) medium: 10 g/L Bactotryptone, 5 g/L Bacto-yeast extract, 10 g/L NaCl, supplemented with 15 g/L agar for plates. 2.2. Transfection in Schneider Cells

1. Schneider’s medium (Gibco). 2. Actin-Gal4 construct (gift from J. Grosshans, Heidelberg, Germany). 3. FuGENE6® transfection reagent (Roche). 4. Rabbit polyclonal antibody against GFP (BioCat).

2.3. Generation of Transgenic Drosophila

1. Incubator for fly cultures with a humidity controller WB750KHF (Mytron, Germany). The humidity was set to 70%. 2. Balancer and driver fly strains (Bloomington Drosophila Stock Center at Indiana University, http://flystocks.bio.indiana. edu/). Balancer flies If/CyO and If/CyO; Sb/TM3Ser (gift from A. M. Voie, Heidelberg, Germany). Driver strains Rh1Gal4 and GMR-Gal4 (gift from C. Desplan, New York, USA). 3. CO2 bottle connected through two different tubings to a glass filter (Neolab 25 209 04) and an injecting needle, respectively, for anaesthetising the flies. 4. Classical stereomicroscope (10× objective) for the observation and sorting of the flies.

2.4. Analysis of Overexpression by Western Blot and Fluorescence Microscopy 2.4.1. Western Blot 2.4.2. Fluorescence Stereomicroscopy

1. Eppendorf homogeniser (Neolab). 2. Classical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (8% separating gel and 3% stacking gel). 3. PVDF membrane (Immobilon-Millipore). 4. Rabbit polyclonal antibody against GFP (BioCat). 1. Stereomicroscope (10× objective) mounted with an HBO mercury lamp and a GFP filter (illumination path BP 480/40 nm, dichroic mirror/reflector 505 nm, observation path LP 510 nm). 2. Fluorescence was recorded with a digital camera (DC200, Leica).

2.5. Flies and Head Harvesting 2.5.1. Fly Food

1. Fly food (see Note 1): Agar (8 g/L), yellow cornmeal (80 g/L), dry yeast (18 g/L), light corn syrup (22 g/L), malt (80 g/L), soybean meal (8 g/L) and the fungicide nipagin

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(0.5 g/L) in tap water (13). All components are boiled for 10 min except nipagin, which has to be added when the mixture is cooled to 60°C. Add propionic acid (7 mL per litre food). 2. Pour the food in small and large vials provided by Greiner BioOne (205101 and 960177, respectively) with, respectively, 10 and 35 mL warm food. 3. After solidification, plugs antimites (25- or 50-mm diameter, respectively; Buddeberg) are set on the tubes. 4. The food is stored at 18°C (see Note 1). 2.5.2. Fly Head Harvesting

1. Microsieve kit for fly head collection (Neolab). 2. Glass beads, 4 mm diameter (Biospec Products).

2.6. Membrane Preparation

1. Glass-to-glass hand homogeniser (20 mL) (Sartorius). 2. Complete® protease inhibitor cocktail (Roche), benzamidine and phenylmethylsulfonylfluoride (PMSF). 3. Sucrose buffer: 50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, 1 mM ethylene glycol-bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid (EGTA), 250 mM sucrose, pH 7.4.

3. Methods Despite the large body of knowledge in the field (14), fly genetics are mostly restricted to specialised laboratories. The generation of transgenic fly strains overexpressing MPs in the eyes for structural studies requires the combination of technical knowledge in two different areas of research: MP engineering and Drosophila genetics. We describe here the overexpression of the human glutamate transporter EAAT2 in the eyes of transgenic flies. The description includes the molecular cloning of the MP into a transposon, the generation of the transgenic fly and the collection of heads for preparation of membranes as a starting material for functional and structural studies. 3.1. Membrane Protein Cloning

pUAST from the pCaSpeR family is a plasmid that is often used for transposition in the Drosophila genome (9). It contains a multiple cloning site (EcoRI-BglII-NotI-EagI-XhoI-KpnIXbaI) for insertion of the gene of interest under the regulation of a Gal4 UAS, followed by the white plus gene (w+), a marker coding for an ABC transporter restoring the natural red colour of the eyes, the P-elements flanking both cistrons and recognised for transposition into the genome and a gene for ampicillin

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Fig. 9.1. (a) Generation of transgenic flies. The gene construct for the membrane protein (MP) to be expressed in fusion with a histidine-tagged GFP (tev: TEV-protease site) was cloned in the multiple cloning site (MCS) of the pUAST plasmid vector, downstream from the regulating element UAS-Gal4. This cistron, together with another one coding for the white gene (w+), a red-eye marker, are included in the transposon delimited by the P-element recognition sites (P ). The pUAST construct was co-injected with a helper plasmid coding for a translocase into the posterior pole of Drosophila embryos. After the usual 12-day cycle of the fly, the survivors were crossed with a white-eye w1118 fly. The red/orange-eye transformants were balanced by successive crossings with If/CyO and If/CyO;Sb/TM3Ser flies. The stable, balanced fly was finally crossed with a driver fly (here GMR-Gal4) for expression into the eyes. (b) The crossing of the UAS-MP-EGFP fly (here UAS-EAAT2-EGFP coding for a fluorescent glutamate transporter) with the driver GMR-Gal4 generated a population of fluorescent flies that could be easily selected using a fluorescence stereomicroscope (10× objective). Left panel a fluorescent eye of a transgenic fly expressing the human EAAT2 in fusion with EGFP. Right panel shows, as a negative control, a transgenic fly that does not have the EGFP transgene.

resistance (Fig. 9.1a) (see Note 2 for sequence and map of pUAST). 1. Amplify the MP gene, in our case the human glutamate transporter EAAT2 fused to EGFP, by classical polymerase chain reaction (PCR) with a high-fidelity polymerase using, for

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instance, 200 ng template, 1 mL each forward and reverse primers (10 mM) (see Note 3), 5 mL polymerase buffer (10X), 1 mL polymerase Expand (3.4 U) and a dNTP (deoxynucleotide 5¢-triphosphate) mixture (25 mM of each four bases) in 50 mL final volume. PCR program: One cycle of 2 min at 95°C, 18 cycles of amplification (95°C for 30 s, 55°C for 30 s, 72°C for 1 min/kbp) and a last cycle of 10 min at 72°C. 2. Analyse the PCR product the same day by electrophoresis in an agarose gel. 3. Ligate 4 mL of PCR product with 1 mL of the pCR2.1 vector provided by the Topo TA Cloning kit. 4. Transform 50 mL chemical-competent Escherichia coli with 5 mL of the ligation mixture by incubation of the mixture 15 min on ice, heat shock for 30 s at 42°C, quick cooling for 2 min on ice and addition of 250 mL LB medium and incubation for 1 h at 37°C in a rolling wheel. 5. Plate the bacteria and select positive clones on kanamycin or ampicillin plates if the template used for the PCR gives ampicillin or kanamycin resistance, respectively. Let grow overnight at 37°C. 6. Inoculate the bacteria clones to test, usually six colonies, in 2 mL LB medium containing kanamycin (3 mg/mL) or ampicillin (10 mg/mL) and let grow overnight at 37°C. 7. Extract and purify the new construct using the classical Miniprep kit (Qiagen) and test the positive clones by digestion of one tenth of the sample by EcoRI. Analyse the restriction products on an agarose gel. 8. Send positive clones for sequencing with the M13(-21) and M13 reverse universal primers annealing on pCR2.1 and internal primers if necessary. 9. The gene is then subcloned into pUAST: Digest 1 mg pUAST vector and 2 mg gene construct in pCR2.1, respectively, with, for instance, EcoRI (20 U) and XbaI (20 U) (see Note 4) in buffer 2 in 30 mL. 10. Purify the linearised vector and the gene construct from an agarose gel using the gel extraction kit. 11. Ligate overnight 10 ng linearised pUAST with the purified gene construct in a stoichiometric ratio of 1:5 (see Note 5) with the T4-DNA ligase (1 mL, 400 U) in a final volume of 30 mL at 18°C. 12. Transform 50 mL chemical-competent E. coli with 10 mL of the ligation mixture by incubation of the mixture for 15 min on ice, heat shock for 30 s at 42°C, quick cooling for 2 min on ice, addition of 250 mL SOC medium and incubation for 1 h at 37°C in a rolling wheel.

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13. Plate the bacteria and select positive clones on an ampicillin plate. Let grow overnight at 37°C. 14. Inoculate the bacteria clones to test, usually 24 colonies, in 5 mL LB medium containing ampicillin (10 mg/mL) and let grow overnight at 37°C. 15. Extract and purify the new construct using the classical Miniprep kit and test the positive clones by double digestion of one fifth of the sample by EcoRI and XbaI. Analyse the restriction products on an agarose gel. 16. Positive constructs are verified for expression in Schneider cells and sequenced (see Note 6) before the injection into fly embryos. 3.2. Transfection in Schneider-2 Cells

The new construct was first tested in a quick transfection experiment. Because the pUAST plasmid has UAS (UAS-Gal4) for regulating the MP expression, a co-transfection with a plasmid offering Gal4 expression (i.e., under the actin promoter) is necessary. 1. Seed S2 Schneider cells at a density of 106 cells per 3-cm Petri dish in 1 mL Schneider cell medium. 2. Transfect the cells after 4 h with a mixture of 0.4 mg of purified actin-Gal4 vector and 0.4 mg of pUAST construct following the instructions for transfection with the FuGENE6 reagent. 3. Analyse protein expression after 48 h by Western blot using, for instance, a commercial antibody directed against the tag or a GFP antibody (see Note 7). 4. If the MP was cloned in frame with the GFP, analyse expression in the cells in vivo by fluorescence microscopy. Typical yields of co-transfection are around 30%.

3.3. Generation of Transgenic Drosophila 3.3.1. Injection of Embryos

3.3.2. Establishing Stable Fly Strains Using Balancer Chromosomes and Chromosome Mapping

The pUAST construct is co-injected with a helper plasmid coding for a transposase into the posterior pole of the embryos of white-eye flies w1118 (Fig. 9.1a). Because this step requires sophisticated material like a microinjection system and some routine of handling (for technical description, see (15)), it is recommended to send the purified pUAST construct (around 30 mg DNA; see Note 8) to a company offering a Drosophila injection service. Why Balance the Flies? In the first transformants (UAS-EAAT2eGFP), meiotic recombination could still occur in females, generating unstable genotypes and possibly different chromosomal location of the transposon. To reduce this effect, the transformants have to be crossed with flies having balancer chromosomes that are rearranged with multiple inversions and cannot undergo transposition events (for more details, see (16)). In the progeny,

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heterozygous flies having one copy of the transposed chromosome and one copy of the balancer chromosome are kept as a stable “balanced stock.” Chromosome Mapping. Chromosome mapping is necessary to know which chromosome received the transposon and needs to be balanced. The chromosome number of Drosophila is 4: a sex chromosome of small size (also called chromosome 1), two large chromosomes 2 and 3, and a small chromosome 4. The probability to obtain a transposition in chromosomes 2 and 3 is higher than for the two other chromosomes. Chromosome mapping is deduced by phenotype exclusion, using the markers carried by the balancer flies. The strain used for balancing over chromosome 2 was If/CyO (see Note 9) (for the nomenclature, see Note 10). The presence of the If marker gives the flies rough eyes, and the Curly O marker CyO gives a curly wings phenotype (14). If in the second offspring of the crossing of the transformant fly with the balancer If/CyO, a fly has all three phenotypes (red eyes, curly wings (CyO phenotype) and rough eyes (If phenotype), the UAS element cannot be on chromosome 2. Therefore, the probability that the element is on chromosome 3 is high. Choice of the Balancer Chromosome. Most of our driver strains carry the Gal4 transposon on chromosome 2. It is therefore ideal to select an UAS-EAAT2-eGFP strain having the UAS element on chromosome 3. The transposed chromosome was then balanced over the TM3 Serrate chromosome (TM3Ser, chromosome 3) using the balancer fly If/CyO; Sb/TM3Ser. The Serrate marker is easy to detect and exhibits a typical wing phenotype (scalloping of the wing margin). The final stable fly stocks have the UAS-EAAT2eGFP/TM3Ser genotype and are ready for further crossing with the driver fly providing Gal4 and induction of expression. Practically (see Fig. 9.1a), 1. Collect If/CyO virgin flies in the early morning plus twice a day from a classical culture of flies grown in large vials. The flies are sorted under a stereomicroscope while anaesthetised under a CO2 stream. A virgin fly can be recognised through different criteria, including the larger size, the black dot visible through the skin of the abdomen, the green meconium presence, pupae-like folded wing (see (16)). The peak of female fertility is between 4 and 15 days after hatching. 2. Select orange- or red-eye transformants (here UAS-EAAT2eGFP) among the male flies delivered by the Drosophila injection service (see Note 11). 3. Cross each male with three virgins of the balancer strain If/ CyO at 25°C. 4. Let flies lay eggs and develop through all stages of development: embryos, first- until third-instar larvae, pupae (life cycle of 12 days).

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5. It is important to remove the parents from the vial before hatching. 6. Collect virgin flies from the balancer strain If/CyO;Sb/ TM3Ser. 7. Collect one male from the progeny of each cross, having red/ orange eyes and the “If” or “CyO” phenotype; cross each with three virgins from the balancer strain If/CyO;Sb/TM3Ser. 8. Remove the parents before hatching. 9. Let the offspring of the first crossing with If/CyO mate again for the chromosome mapping. 10. From the progeny of the crossing with balancer If/CyO;Sb/ TM3Ser, cross a red-eye virgin of phenotype CyO, TM3Ser, not If because of the eye phenotype, with a red-eye male of the same phenotype. The progeny of this crossing are balanced and stable and can be stored at 18°C. The establishment of a new stable fly strain takes around 2 months (see Note 12). If the UAS element was mapped on chromosome 3, the expected genotype is most likely: +;

CyO UAS ; ;+ + TM3

If the UAS element was mapped on chromosome 2, the expected genotype is most likely: +;

3.3.3. Choice of the Driver

UAS TM3 ; ;+ CyO +

The balanced fly is still not expressing the recombinant MP because of the lack of the transcription factor Gal4. It requires crossing with a so-called driver, a fly expressing Gal4 under the control of a specific promoter. There are a number of different eye drivers that can be used that are available in the Bloomington Drosophila Stock Center. The “glass multiple reporter” GMR was used in this study for its high Gal4 induction and therefore high MP expression. 1. Collect virgin flies from the driver strain GMR-Gal4. 2. Cross a male of the new stable UAS strain balanced over TM3Ser (chromosome 3) with three virgin fly homozygotes for GMR-Gal4 on chromosome 2 (see Note 13). 3. Remove the parents before hatching. 4. From the new TM3Ser progeny, recross flies having the phenotype CyO, not Ser/TM3, and select the best fluorescent offspring for obtaining a homozygous population (see Note 14).

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3.4. Analysis of Overexpression by Western Blot and In Vivo Fluorescence Microscopy

The transgenic flies expressing an MP fused with EGFP can be analysed in Western blot and fluorescence microscopy. For the Western blot analysis, five heads are dissected and homogenised using an Eppendorf homogeniser in 30 mL loading buffer. The samples are analysed as described for the Schneider cell expression. To test the overall fluorescence of the eyes (Fig. 9.1b) and selecting the best-expressing fly, a mercury fluorescence lamp and a GFP filter simply adapted on a classical stereomicroscope are required, which should be affordable for most laboratories.

3.5. Flies and Heads Harvesting

Flies are reared in small vials at 18°C for stocks and 25°C for crossings. Large vials are used for scaling-up the cultures, and flies are grown at room temperature (22°C). For large cultures (milligram amounts of target MP), 100 large vials distributed in three racks (43 × 30cm) give 50 mL flies per week. Flies are put to sleep by CO2 anaesthesia and collected in liquid nitrogen. The vials can be reused two or three times. New crossings were performed every 3 months to avoid overgrowth by contaminating flies.

3.5.1. Fly Cultures

3.5.2. Harvesting the Heads

1. Cool in liquid nitrogen a 5-mL volume of glass beads, the set of sieves, a funnel small enough to be adapted on a 15-mL Falcon™ tube and a 15-mL Falcon tube. 2. Transfer the fly stock from the −80°C freezer into liquid nitrogen. 3. Mix the 5 mL frozen beads with a 10-mL volume of flies in a cold 50-mL Falcon tube and shake vigorously three times to get rid of the wings that remain sticking to the Falcon tube. 4. Mount rapidly the sieve kit on the bench. Take the three smallest of the five sieves of the kit and mount a triple-sieve stack arranged in downward decreasing mesh diameters. 5. Transfer rapidly the mixture of flies and beads on the top of the cold triple-sieve stack. 6. Shake the sieve stack to break the fragile neck of the frozen flies. 7. Mount quickly the 15-mL Falcon tube on ice with the cold funnel on top. 8. Harvest the heads retained on the middle sieve by pouring them in the funnel (see Note 15). 9. Transfer the triple-sieve stack, the funnel and the 15-mL Falcon tube containing the heads back into liquid nitrogen. 10. Start again from the beginning with the next 10 mL of frozen flies (see Note 16). 11. Store the heads at −80°C.

3.6. Membrane Preparation

1. Pour a volume of 1 mL frozen heads into a 20-mL glass-toglass hand homogeniser (see Note 16).

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2. Because fly heads float in liquid, first give a small volume of 200 mL ice-cold sucrose buffer with protease inhibitors (Complete, 1 mM benzamidine, 1 mM PMSF) and remove air bubbles by simple pressure of the piston. 3. Add 1 mL ice-cold buffer to the compact tissue and homogenise the heads. 4. Centrifuge the homogenised material for 10 min at 2,000g, 4°C, to pellet cuticles and store the supernatant on ice. 5. Resuspend the pellet in 500 mL sucrose buffer and rehomogenise. 6. Repeat the procedure until the supernatant of centrifugation does not contain membranes, around three times. 7. Pool supernatants and centrifuge membranes for 60 min at 70,000g, 4°C. 8. Resuspend the resulting pellet in sucrose buffer at a concentration of 10 mg protein/millilitre and store in aliquots at −80°C. The membranes are ready for protein purification or activity tests (the MP analysis and purification are described in (17)).

4. Notes 1. The quality of the fly food is important, especially optimising the water content: If the food is too wet, the flies will stick and die; if the food is too dry, the culture will give low yields of larvae. The fly food must preferably be consumed in less than 3 weeks to avoid fungi growing and dryness. 2. The correct sequence of pUAST is available from Brand’s group (http://www.gurdon.cam.ac.uk/~brandlab/). The sequence from other Web sites may be incorrect. 3. The forward primer is designed with a Kozak-like sequence “cacaag” or “cggagc” directly preceding the start codon (18). Depending on the MP, different tags will be designed in the forward or reverse primer. For histidine, depending on the construct, the tag can be elongated from 6 to 8 or 10 histidines if necessary. 4. Unmethylated pUAST is necessary for optimal restriction with XbaI. 5. Different stoichiometric ratios of the gene construct and pUAST vector should be tried in case of tricky ligation. 6. The following sequencing primers matching with pUAST were routinely used: forward 5¢-CGTCAATTCAATTCAAA

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CAAGC (−196 upstream EcoRI) and the reverse 5¢-GAGCTTT AAATCTCTGTAGG (−53 downstream XbaI). 7. For Western blot, MPs are not boiled but prepared 30 min at 45°C in a classical loading buffer. The antibody against GFP gave better results if incubated overnight. 8. pUAST is a low -copy plasmid and a midi-preparation (100 mL of bacteria culture) will be necessary for Schneider cell tests and fly generation. 9. Balancer fly strains are available in the Bloomington Drosophila Stock Center at Indiana University (http://flystocks.bio. indiana.edu/). 10. The standard nomenclature in Fly Genetics utilises a “+” for wild type, the four chromosomes are separated by a “;” (i.e., a wild-type fly would be +;+;+;+). The two alleles of sister chromosomes are separated by a “/” or “_” (i.e., If/CyO is the name of a fly having a balancer chromosome 2 “If” and a sister balancer chromosome 2 “CyO”). For ease of writing, wild-type chromosomes 1 and 4 of the strain If/CyO;Sb/ TM3Ser (+;If/CyO;Sb/TM3Ser;+) have been omitted. 11. To obtain additional fly strains per construct, the females can also be used and crossed with males of the balancer fly. 12. Already balanced fly strains can be ordered in from Drosophila injection services to avoid this time-consuming task that demands some experience in fly genetics. 13. Sometimes, it is advisable to use a weaker promoter (e.g., when protein expression is toxic). The rhodopsin promoter Rh1-Gal4 was successfully tested as a driver (e.g., for the human vasopressin receptor V1aR) (11). 14. The homozygous CyO genotype is lethal. 15. The top sieve with 25 mesh retained both the thorax-abdomen parts and the glass beads but not the heads, and the sieve with 35 mesh retained the heads but not the legs and wings. 16. A 50-mL volume of flies represents around 10,000 flies and gives 1 g of heads or 20 mg of membranes.

Acknowledgments We thank Ann Mari Voie (EMBL, Heidelberg) for technical advices and Silke Adrian for excellent technical assistance. This work was supported by the European Community Specific Targeted Research Project grant IMPS (Innovative Tools for Membrane Structural Proteomics, FP6-2003-LifeSciHealth 513770).

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References 1. Grisshammer R, Tate CG (1995) Overexpression of integral membrane proteins for structural studies. Q Rev Biophys 28:315–422 2. Tate CG (2001) Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett 504:94–98 3. Grisshammer R (2006) Understanding recombinant expression of membrane proteins. Curr Opin Biotechnol 17:337–340 4. Junge F, Schneider B, Reckel S, Schwarz D, Dotsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol Life Sci 65:1729–1755 5. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE et al (2000) Crystal structure of rhodopsin: a G proteincoupled receptor. Science 289:739–745 6. Zuker CS (1996) The biology of vision of Drosophila. Proc Natl Acad Sci U S A 93: 571–576 7. Fotiadis D, Jastrzebska B, Philippsen A, Muller DJ, Palczewski K, Engel A (2006) Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors. Curr Opin Struct Biol 16:252–259 8. Kock I, Bulgakova NA, Knust E, Sinning I, Panneels V (2009) Targeting of Drosophila rhodopsin requires helix 8 but not the distal C-terminus. PLoS One 4(7):e6101 9. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415

10. Elliott DA, Brand AH (2008) The GAL4 system: a versatile system for the expression of genes. Methods Mol Biol 420:79–95 11. Eroglu C, Cronet P, Panneels V, Beaufils P, Sinning I (2002) Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye. EMBO Rep 3: 491–496 12. Eroglu C, Brugger B, Wieland F, Sinning I (2003) Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts. Proc Natl Acad Sci U S A 100:10219–10224 13. Roberts DB (1998) Drosophila: a practical approach. Oxford University Press, Oxford 14. Lindsley DL, Zimm GG (1992) The genome of Drosophila melanogaster. Academic, San Diego, CA 15. Voie AM, Cohen S (1998) Cell biology: a laboratory handbook, vol. 3. Academic, New York, pp 510–517 16. Ashburner M (1989) Drosophila: a laboratory handbook and manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 17. Panneels V, Eroglu C, Cronet P, Sinning I (2003) Pharmacological characterization and immunoaffinity purification of metabotropic glutamate receptor from Drosophila overexpressed in Sf9 cells. Protein Exp Purif 30: 275–282 18. Cavener DR (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res 15:1353–1361

Chapter 10 Expression of Mammalian Membrane Proteins in Mammalian Cells Using Semliki Forest Virus Vectors Kenneth Lundstrom Abstract One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation and bacterial systems to expression in animal cells. In contrast to soluble proteins, the expression levels have been relatively low due to the following reasons: The topology of membrane proteins requires special, posttranslational processing, folding, and insertion into membranes, which often are mammalian cell specific. Despite these strict demands, functional membrane proteins (G protein-coupled receptors, ion channels, and transporters) have been successfully expressed in bacterial, yeast, and insect cells. A general drawback observed in prokaryotic cells is that accumulation of foreign protein in membranes is toxic and results in growth arrest and therefore low yields of recombinant protein. In this chapter, the focus is on expression of recombinant mammalian membrane proteins in mammalian host cells, particularly applying Semliki Forest virus (SFV) vectors. Replication-deficient SFV vectors are rapidly generated at high titers in BHK-21 (Baby Hamster Kidney) cells, which then are applied for a broad range of mammalian and nonmammalian cells. The SFV system has provided high expression levels of topologically different proteins, especially for membrane proteins. Robust ligand-binding assays and functional coupling to G proteins and electrophysiological recordings have made the SFV system an attractive tool in drug discovery. Furthermore, the high susceptibility of SFV vectors to primary neurons has allowed various applications in neuroscience. Establishment of large-scale production in mammalian adherent and suspension cultures has allowed production of hundreds of milligrams of membrane proteins that has allowed their submission to serious structural biology approaches. In this context, a structural genomics program for SFVbased overexpression of 100 GPCRs was established. Key words: Large-scale production, mammalian cells, Semliki forest virus, structural biology

1. Introduction Semliki Forest virus (SFV) is a single-stranded RNA (ss-RNA) alphavirus with an envelope structure belonging to the Togaviridae family (1). The SFV system exists in different variations, namely, I. Mus-Veteau (ed.), Heterologous Expression of Membrane Proteins, Methods in Molecular Biology, vol. 601 DOI 10.1007/978-1-60761-344-2_10, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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replication-deficient, replication-competent, and DNA-based vectors (2, 3). Here, the focus is only on replication-deficient vectors as these are best suited for recombinant protein expression in mammalian cell lines. The gene of interest is cloned into the SFV expression vector downstream of the highly efficient subgenomic SFV 26S promoter (Fig. 10.1). Next, RNA is in vitro transcribed from the expression vector and cotransfected into BHK-21 (Baby Hamster Kidney cells) together with RNA coding for the structural proteins (capsid and envelope proteins). Due to the presence of the packaging signal only located on the recombinant RNA originating from the expression vector, the viral progeny will only contain this type of RNA (Fig. 10.2). The lack of RNA coding for the structural proteins renders the recombinant SFV particles replication deficient. However, the introduction of the recombinant RNA including the SFV nonstructural replicase genes on infection results in highly efficient RNA replication (estimated to 200,000 copies per cell), which is the basis for the high level of heterologous gene expression. Moreover, the SFV infection results in an almost-complete shutdown of endogenous gene expression, which further contributes to the almost-unique production of recombinant protein. The SFV system has found its solid place due to the following reasons: The SFV expression vector can accommodate at least 6-kb inserts (4); virus stocks can be prepared in less than 2 days, resulting in titers of 109 to 1010 infectious particles per milliliter. The broad host range of SFV allows transduction of a large number of mammalian cell lines and primary cell cultures (5). Expression of G protein-coupled receptors (GPCRs) and ion channels from SFV vectors has provided pharmacologically functional membrane proteins (6) and has demonstrated coupling of G proteins to GPCRs (7) and electrophysiological responses of ion channels (8). When coexpression of several genes is requested, for instance, G proteins and GPCRs, multiple SFV vectors can be coinfected (9). Efficient infection of both adherent and suspension cultures of mammalian cells has allowed scale-up of recombinant protein production in spinner and roller flasks as well as in bioreactors, which has allowed the production of hundreds of milligrams of recombinant membrane proteins (10). This development has allowed the application of SFV for structural genomics initiatives on membrane proteins (11, 12). The disadvantage of applying the SFV system is that, especially for large-scale production, the virus stock production is relatively expensive. The high costs are related to the in vitro transcription process, which requires expensive reagents such as (m7G(5') ppp(5')G) CAP analogue and SP6 RNA polymerase. Attempts to facilitate and reduce the costs for virus stock preparation have been introduced by the development of alphavirus packaging cell lines (13). Another concern has been the safety aspects of using

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SFV, especially for large-scale production, which obviously requires large volumes of virus. This aspect has been thoroughly investigated (14), and there is strong confidence in the safe use of SFV vectors. In this chapter, the subcloning into SFV vectors, recombinant virus production, and application of SFV particles for expression of membrane proteins in mammalian cell lines are described.

2. Materials 2.1. Cell Cultures

1. BHK-21 (Baby Hamster Kidney) cells.

2.1.1. Adherent Cells

2. CHO-K1 (Chinese Hamster Ovary) cells. 3. HEK293 (Human Embryonic Kidney) cells. BHK-21 cells are commonly used host cells for the packaging of recombinant SFV particles. They can be cultured in various media, but a 1:1 mixture of Dulbecco’s modified F-12 medium (Gibco BRL) and Iscove’s modified Dulbecco’s medium (IMDM) (Gibco BRL) supplemented with 4 mM glutamate and 10% fetal calf serum seemed to give robust cell growth and high titer virus stock production. CHO-K1 and HEK293 cells were cultured in the same medium. Application of alternative cell lines may require special media.

2.2. Suspension Cultures

2.3. SFV and Other Plasmid Vectors

1. DHI is a combination of DME, HamF12 and IMDM media 2. HL medium. Suspension cultures of BHK-21 and CHO-K1 cells for large-scale protein production were established in DHI medium: a mixture of DME, Ham F-12 and IMDM (Iscore’s Modified Dublecco’s Medium) media (1:1:2) supplemented with 5 mM glutamine, 5 g/L glucose, 5 mg/L insulin, 6 mg/L transferrin, 0.25% (v/v) Primatone RL (Sheffield Products/Quest International), 0.01% (v/v) Synperonic F68 (Serva), 20 mM selenite, 20 mM ethanol amine, and 2.5 mM β-mercaptoethanol (15). HEK293 suspension cultures were grown in HL medium: A 2:1 (by weight) mixture of DHI and RPMI-1640 supplemented with 5 mM glutamine and 5 g/L glucose (16). A higher concentration of 15 mg/L of transferrin was used for HEK293 cells. Otherwise, the components were identical to those used for BHK-21 and CHO-K1 cells. For SFV-based expression, a two-vector system for recombinant particle production was applied (Fig. 10.1). The most commonly used SFV vectors are pSFV1 and pSFV2gen (also called pSFV4.2).

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a

1106 Bcl I 1425 Bpl I 1463 Stu I 1636 Ssi I 1799 Ecl 136II 1799 Sac I

Xmn I 9507 Pvu I 9279

pSFVgen2 Bsp LU11I 8019 Sap I 7896 Nru I 7830 Bsp 120I Apa I Avr II Bst BI Not I Spe I Xho I Sci I Xma I Sma I Bss HII Rsr II Bam HI

7484 7484 7469 7463 7454 7441 7432 7432 7424 7424 7415 7406 7400 Bgl II 6713 Xba I 6638

10610 base pairs Unique Sites

4916 Bsu 36I

Age I 5372

Fig. 10.1. Maps of SFV expression and helper vectors. (a) pSFV2gen; (b) pSFV-Helper2.

These vectors are linearized by SpeI and NruI, respectively. Both vectors can also be cut with SapI. SpeI is used for the linearization of the pSFV-helper2 vector. Polymase chain reaction (PCR) frag-

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ments are preferentially cloned into the pCR4Blunt-TOPO vector (Invitrogen) prior to subcloning into SFV vectors. 2.4. Reagents and Equipment

1. Restriction enzymes: NruI, SapI, and SpeI. 2. Agarose gel: 0.8%. 3. Gel electrophoresis apparatus. 4. Phenol/chloroform/isoamylalcohol: 25:24:1 (v/v/v). 5. 3M sodium actetate, pH 4.8. 6. Ethanol, 70% and 95% (v/v). 7. MicroSpin™ S-200 HR columns (Amersham). 8. SP6 buffer (10X): 400 mM HEPES, pH 7.4, 60 mM magnesium acetate, 20 mM spermidine. 9. 50 mM dithiotheritol (DTT). 10. rNTP mix: 10 mM rATP, 10 mM rCTP, 10 mM rUTP, 5 mM rGTP. 11. m7G(5¢)ppp(5¢)G CAP analogue. 12. RNase (ribonuclease) inhibitor: 10–50 U/mL. 13. SP6 RNA polymerase: 10–20 U/mL. 14. Trypsin–ethylenediaminetetraacetic acid (EDTA): 0.25% trypsin, 1 mM EDTA × 4 Na. 15. Phosphate-buffered saline (PBS). 16. Microcentrifuge, 1.5-mL microcentrifuge tubes. 17. Heating blocks. 18. Water baths. 19. Electroporator. 20. Electroporation cuvettes: 0.2 and 0.4 cm. 21. Tissue culture flasks: T25, T75, and T175. 22. Microwell plates: 6-, 12-, and 24-well plates. 23. Falcon tubes: 15 and 50 mL. 24. Sterile syringes: 1, 10, and 50 mL. 25. Sterile filters: 0.22 mm. 26. Opti-MEM I reduced-serum medium (Gibco BRL). 27. X-gal stock solutions: 50 mM K ferricyanide (+4°C); 50 mM K ferrocyanide (+4°C); 1M MgCl (room temperature); 2% X-gal in dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) (−20°C). 28. X-gal staining solution: 1X PBS (1/10 of stock), 5 mM K ferricyanide (1/10 of stock), 5 mM K ferrocyanide (1/10 of stock), 2 mM MgCl (1/500 of stock), 1 mg/ml X-gal (1/20 of stock).

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29. Moviol 4–88 containing 2.5% DABCO (1,4-diazobicyclo(2.2.2)-octane). 30. Lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Nonidet P-40 (NP40)). 31. Hybond ECL nitrocellulose filter (Amersham). 32. TBST (TBS (Tris-based saline) buffer with 0.1% Tween-20). 33. ECL Chemiluminescence kit (Amersham). 34. Starvation medium (methionine-free MEM (Minimal Essential Medium), 2 mM glutamine, 20 mM HEPES). 35. Chase medium (E-MEM, 2 mM glutamine, 20 mM HEPES, 150 mg/mL unlabeled methionine).

3. Methods 3.1. Subcloning into SFV Vectors

General cloning procedures are performed for the subcloning of genes of interest into the multiple cloning sites (MCSs) of the SFV expression vectors. Due to the large size of the SFV vectors, it is advisable initially to clone PCR fragments into other types of cloning vectors, such as the pCR4Blunt-TOPO vector (Invitrogen). Please note that the linearization sites (SpeI, SapI, and NruI) in SFV cannot be used as cloning sites as the region between the MCS and the linearization sites contains the RNA replication and polyA+ signals. The unique XmnI site (at position 9929) in the pSFV1 vector cannot be used as the RNA transcript becomes too long to function properly. The presence of inserts and their correct orientation can be analyzed by restriction endonuclease digestions and nucleotide sequencing. DNA templates for in vitro transcription reactions should preferentially be of DNA Midiprep or Maxiprep quality. However, initial tests can be carried out with Miniprep DNA (see Note 1).

3.2. DNA Linearization

SFV plasmid vectors are linearized by SpeI, SapI, or NruI under standard restriction digestion conditions. The completion of linearization should be verified by agarose gel electrophoresis (0.8%). 1. Linearize at least 5–10 mg plasmid DNA (larger quantities can be stored at −20°C) 2. Confirm complete digestion by agarose gel electrophoresis in comparison to uncut plasmid. 3. Purify linearized DNA by phenol/chloroform extraction followed by ethanol precipitation (overnight at −20°C or 15 min at −80°C) or on MicroSpin S-200 HR columns according to the manufacturer’s instructions.

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4. In case of phenol/chloroform extraction, centrifuge ethanol precipitates for 15 min at 18,000g at +4°C and wash with 70% ethanol.

3.3. In Vitro Transcription

Efficient in vitro RNA transcription is essential for the production of high-titer virus stocks (Fig. 10.2). Preferentially, fresh RNA preparations are made for each electroporation, although RNA transcripts can be stored for shorter periods (weeks) at −80°C. In case large quantities of virus stocks are produced, the process can be scaled up by multiplying the volumes for the in vitro transcription reactions and performing multiple electroporations in parallel. Prepare the in vitro transcription reactions at room temperature as the SP6 buffer contains spermidine, which might lead to precipitation at lower temperatures. Add the enzyme components last. Set up separate in vitro transcription reactions for expression and helper vectors in sterile 1.5-mL microcentrifuge tubes (see Note 2).

3.3.1. SFV In Vitro Transcription Reaction

5 mL (2.5 mg) linearized plasmid DNA 5 mL 10X SP6 buffer 5 mL 10 mM m7G(5¢)ppp(5¢)G

In฀vitr o฀ transcription฀

Electroporation฀

BHK฀cells฀

rSFV฀parlticles฀

recombinant฀฀ protein฀

Fig. 10.2. Schematic presentation of SFV particle production.

B & W IN PRINT

5. Repeat centrifugation for 5 min, air dry or lyophilize the DNA pellet, and resuspend in RNase-free H2O at a final concentration of 0.5 mg/mL.

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5 mL 50 mM DTT 5 mL rNTP mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, 5 mM rGTP) x mL RNase-free H2O to reach a final volume of 50 mL 1.5 mL (50 U/mL) RNase Inhibitor mL (20 U/mL) SP6 RNA polymerase 1. Mix all reaction components and spin briefly in a microcentrifuge. 2. Incubate 1 h at 37°C (see Note 3). 3. Load 1- to 4-mL aliquots on a 0.8% agarose gel for RNA quality control and continue the incubation for the rest of the samples. High-quality RNA generates relatively thick bands and no smearing. The size of ss-RNA molecules cannot be directly compared to DNA markers (l DNA or DNA ladders) as ss-RNA has four times faster mobility, but RNA from expression vectors has an approximate (depending on insert size) mobility of 8 kb, whereas helper RNA runs faster. 4. RNA molecules can be directly subjected to electroporations or stored at −80°C for later use. In case of storage, it is essential to reevaluate the RNA quality on thawing. Each transcription reaction should generate approximately 20–50 mg of RNA. 3.4. Electroporation of RNA

BHK-21 cells are good producers of high-titer SFV stocks, although other host cell can be used. The cells should not be passaged for more than 3 months as they lose their viability. Moreover, cells should not be cultured for more than 48 h prior to electroporation and should not exceed more than 80% confluency as old and too dense cell cultures generate significantly lower virus titers (see Note 4). 1. Plate 1:3 (overnight) or 1:5 (over two nights) sufficient quantities of BHK-21 cells in T175 flask(s). 2. Confirm cell growth and morphology by microscopy. 3. Wash cells once with PBS and trypsinize with 6 mL trypsinEDTA per T175 flask for 5 min at 37°C. 4. Resuspend cells to remove clumps and add cell culture medium to 25 mL. 5. Centrifuge for 5 min at 800g. 6. Resuspend cell pellet in a small volume (