Production of Membrane Proteins: Strategies for Expression and Isolation

Membrane proteins represent the largest class of proteins in the human genome, and are targets for a majority of all currently marketed therapeutic agents, as well as ongoing drug discovery efforts. Efficient and reliable production of these proteins is critical for high–throughput screening applications, drug discovery and design, structural biology, and biochemical and biophysical analysis of structure and function.
Designed as a research–level guide to current strategies and methods of membrane protein production on the small to intermediate scale, this practice–oriented book provides detailed, step–by–step laboratory protocols as well as an explanation of the principles behind each method, together with a discussion of its relative advantages and disadvantages.
Following an introductory section on current challenges in membrane protein production, the book goes on to look at expression systems, emerging methods and approaches, and protein specific considerations. Case studies illustrate how to select or sample the optimal production system for any desired membrane protein, saving both time and money on the laboratory as well as the technical production scale. Unique in its coverage of "difficult" proteins with large membrane–embedded domains, proteins from extremophiles, peripheral membrane proteins, and protein fragments, this is essential reading for biochemists, molecular biologists, and biotechnologists, as well as those working in the biotechnological and pharmaceutical industries.

Spis treści:
Preface XV
List of Contributors XVII
Introduction 1
Anne Skaja Robinson and Patrick J. Loll
Expression 2
Solubilization and Structural Methods 5
Abbreviations 7
References 7
Part One Expression Systems 11
1 Bacterial Systems 13
James Samuelson
1.1 Introduction 13
1.2 Understanding the Problem 14
1.3 Vector/Promoter Types 15
1.4 T7 Expression System 20
1.5 Tunable T7 Expression Systems 21
1.6 Other Useful Membrane Protein Expression Strains 22
1.7 Clone Stability 23
1.8 Media Types 24
1.9 Fusion Partners/Membrane Targeting Peptides 25
1.10 Chaperone Overexpression 26
1.11 Cautionary Notes Related to Chaperone Overexpression 27
1.12 Emerging Role of Quality Control Proteases 27
1.13 Tag Selection 28
1.14 Potential Expression Yield 29
1.15 Strategies to Overcome Protein Instability 30
Acknowledgments 31
Abbreviations 31
References 31
2 Membrane Protein Expression in Saccharomyces cerevisiae 37
Zachary Britton, Carissa Young, Özge Can, Patrick McNeely, Andrea Naranjo, and Anne Skaja Robinson
2.1 Introduction 37
2.2 Getting Started 38
2.2.1 Promoter Systems 38
2.2.1.1 Constitutive Promoters 38
2.2.1.2 Inducible Promoters 38
2.2.2 Host Strains, Selection Strategies, and Plasmids 39
2.2.2.1 Host Strains 39
2.2.2.2 Selection Strategies 40
2.2.2.3 Plasmids and Homologous Recombination 40
2.2.3 Expression Conditions 42
2.3 Special Considerations 43
2.3.1 Post–Translational Modifications 43
2.3.1.1 Glycosylation 43
2.3.1.2 Disulfide Bond Formation 44
2.3.2 Lipid Requirements 44
2.3.2.1 Glycerophospholipids 45
2.3.2.2 Sphingolipids 46
2.3.2.3 Sterols 46
2.3.3 Signal Sequences 47
2.3.4 Topology 47
2.3.5 Cellular Responses to Membrane Protein Expression 49
2.3.5.1 UPR 49
2.3.5.2 HSR 49
2.4 Case Studies 49
2.4.1 Ste2p 50
2.4.2 Pma1p 50
2.4.3 CFTR 60
2.5 Conclusion s 61
Abbreviations 62
References 62
3 Expression Systems: Pichia pastoris 75
Fatima Alkhalfi oui, Christel Logez, Olivier Bornert, and Renaud Wagner
3.1 Introduction 75
3.2 A (Brief) Summary on the (Long) History of P. pastoris 75
3.3 Introducing P. pastoris as a Biotechnological Tool: Its (Extended) Strengths and (Limited) Weaknesses 76
3.4 Basics of the P. pastoris Expression System 77
3.4.1 Methanol Utilization Pathway 77
3.4.2 Host Strains and Plasmids 78
3.4.3 Transformation and Clone Selection Strategies 80
3.4.4 Expression Conditions and Culturing Formats 80
3.5 Successful Large–Scale Expression of Membrane Proteins Using P. pastoris 81
3.5.1 P. pastoris for Membrane Protein Expression 81
3.5.2 Common Trends for an Efficient Expression of Membrane Proteins in P. pastoris 92
3.6 Guidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as Models 94
3.6.1 Design and Selection of Enhanced Expression Clones 95
3.6.2 Optimization of the Expression Conditions 96
3.6.3 Yeast Cell Lysis 98
3.7 Conclusions and Future Directions 99
Acknowledgments 99
Abbreviations 99
References 100
4 Heterologous Production of Active Mammalian G–Protein–Coupled Receptors Using Baculovirus–Infected Insect Cells 109
Mark Chiu, Brian Estvander, Timothy Esbenshade, Steve Kakavas, Kathy Krueger, Marc Lake, and Ana Pereda–Lopez
4.1 Introduction 109
4.2 Experimental 113
4.2.1 Generation of Recombinant Baculovirus 113
4.2.2 Baculovirus Infection of Insect Cells 115
4.2.3 Case Study: Histamine H3 Receptor 118
4.2.3.1 Solubilization of the Histamine H3 Receptor 125
4.2.3.2 Assay Validation 125< br>4.2.3.3 Competition Analysis of Solubilized versus Membrane–Bound Receptor 126
4.3 Conclusions and Future Perspectives 128
4.3.1 Executive Summary 130
4.3.2 Future Perspectives 130
Abbreviation 131
References 131
5 Membrane Protein Expression in Mammalian Cells 139
Deniz B. Hizal, Erika Ohsfeldt, Sunny Mai, and Michael J. Betenbaugh
5.1 Introduction 139
5.2 Mammalian Systems 140
5.2.1 Cell Culture Types and Media Optimization 140
5.2.1.1 Adherent Cell Culture 140
5.2.1.2 Suspension Cell Culture 141
5.2.1.3 Batch and Fed–Batch Culture 141
5.2.1.4 Perfusion Process 141
5.2.1.5 Media Optimization 141
5.2.2 Gene Delivery and Expression in Mammalian Systems 143
5.2.2.1 High Transfection Efficiency in Adherent Cell Cultures with Cationic Liposome 143
5.2.3 Post–Translational Modifications in Mammalian Systems 147
5.2.3.1 Glycosylation 148
5.2.3.2 Protein Lipidation 149
5.3 Case Studies 150
5.3.1 Increasing Membrane Protein Expression by Virus Vectors 150
5.3.2 Anti–apoptosis Engineering for Increasing Membrane Protein Expression 152
5.3.3 Increasing Membrane Protein Expression by Chaperones 156
5.3.4 Membrane Protein Expression in Cancer Cell Lines 157
5.3.5 Membrane Proteins as Biotherapeutics 158
5.4 Conclusions 159
Abbreviations 160
References 161
6 Membrane Protein Production Using Photosynthetic Bacteria: A Practical Guide 167
Philip D. Laible, Donna L. Mielke, and Deborah K. Hanson
6.1 Introduction 167
6.1.1 The Membrane Protein Problem 167
6.1.2 Exploiting the Physiology of Photosynthetic Bacteria 168
6.1.3 Expression Strategies 170
6.1.3.1 Design of the Expression Plasmids 170
6.1.3.2 Design of Expression Hosts 172
6.1.3.3 Autoinduction Conditions 173
6.1.4 Summary of Success Stories 174
6.2 Preparation of Expression Constructs 175
6.2.1 Platform Vector Preparation 175
6.2.1.1 Large–Scale Vector Preparation Protocol for Ligation–Dependent Cloning 175
6.2.1.2 Large–Scale Vector Preparation Protocol for LIC 176
6.2.2 Design of Oligonucleotide Primers for Gene Amplification and Cloning 177
6.2.2.1 Ligation–Dependent Cloning 177
6.2.2.2 LIC 177
6.2.3 Target Gene Preparation 178
6.2.3.1 PCR Amplification of Target Gene 178
6.2.3.2 Restriction Enzyme Digestion of PCR Amplicon 178
6.2.3.3 Digestion of PCR Amplicon to Generate LIC Overhangs 178
6.2.4 Cloning of Digested Amplicons 178
6.2.4.1 Generation of Recombinant Plasmids 178
6.2.4.2 Transformation of E. coli with Ligation or LIC Reactions 179
6.2.5 Screening for Successful Insertion of Target Gene into Platform Vector 179
6.3 Transfer of Plasmid DNA to Rhodobacter via Conjugal Mating 180
6.3.1 Transformation of E. coli S17–1 180
6.3.2 Conjugation of E. coli with R. sphaeroides 180
6.4 Small–Scale Screening for Expression and Localization of Target Protein in Rhodobacter 181
6.4.1 Small–Scale Growth and Preparation of Samples for SDS–PAGE 182
6.4.1.1 Growth and Harvest of Expression Strains 182
6.4.1.2 Preparing Whole–Cell Samples for SDS–PAGE 183
6.4.1.3 Preparing Membranes and the Soluble Fraction for SDS–PAGE 184
6.4.2 SDS–PAGE Followed by Electroblotting of Proteins to PVDF Membrane 185
6.4.3 Immunoblot Development 186
6.5 Large–Scale Culture 187
6.5.1 Growth and Harvest of Expression Culture 187
6.5.2 Cell Lysis 188
6.5.3 Membrane Isolation 188
6.6 Detergent Solubilization and Chromatographic Purification of Expressed Membrane Proteins 189
6.6.1 Solubilization of Membrane Proteins 190
6.6.2 Chromatography 190
6.6.2.1 Bench–Top Affinity Chromatography 190
6.6.2.2 Affinity Chromatography Using an ÄKTA–FPLC™ 191
6.7 Protein Identification and Assessment of Purity 192
6.8 Preparations of Specialized Rhodobacter Membranes 192
Appendix: Media and Buffer Formulations 194
Abbreviations 196
References 197
Part Two Protein–Specific Considerations 199
7 Peripheral Membrane Protein Production for Structural and Functional Studies 201
Brian J. Bahnson
7.1 Introduction 201
7.2 Case Studies of Peripheral Membrane Proteins 204
7.2.1 Electrostatic Interactions 204
7.2.1.1 Case 1: Cytochrome c2 205
7.2.1.2 Case 2: Group IB Secreted Phospholipase A2 205
7.2.2 Hydrophobic Patch 207
7.2.2.1 Case 1: Plasma Platelet–Activating Factor Acetylhydrolase 208
7.2.2.2 Case 2: Human Serum Paraoxonase 1 210
7.2.3 Covalent Lipid Anchor 210
7.2.3.1 Case 1: Recoverin 211
7.2.3.2 Case 2: Intracellular Platelet–Activating Factor Acetylhydrolase Type II 212
7.2.4 Case 3: Palmitoylation of Human Proteins in Cell Culture 212
7.2.5 Lipid–Binding Domain 212
7.2.5.1 Case 1: Pleckstrin Homology Domain 213
7.2.5.2 Case 2: C2 Domain 213
7.3 Conclusions 214
Acknowledgments 215
Abbreviations 215
References 215
8 Expression of G–Protein–Coupled Receptors 219
Alexei Yeliseev and Krishna Vukoti
8.1 Introduction 219
8.2 Bacterial Expression of GPC Rs 220
8.3 Expression of GPCRs in Inclusion Bodies, and Refolding 228
8.4 Expression of GPCRs in Yeast 229
8.5 Expression of GPCRs in Insect Cells 231
8.6 Expression of GPCRs in Mammalian Cell Lines 234
8.7 Expression of GPCRs in Retina Rod Cells 234
8.8 Expression of GPCRs in a Cell–Free System 235
8.9 Stabilization of GPCRs during Solubilization and Purification 238
8.10 Conclusions 238
Acknowledgments 239
Abbreviations 239
References 240
9 Structural Biology of Membrane Proteins 249
David Salom and Krzysztof Palczewski
9.1 Introduction 249
9.2 Folding and Structural Analysis of Membrane Proteins 249
9.2.1 Folding 249
9.2.2 Prediction Methods 251
9.2.3 Membrane Insertion 251
9.2.4 Estimating the Molecular Weight of Membrane Proteins 252
9.2.5 Amino Acid Composition 252
9.2.6 Transmembrane Helix Association Motifs and Membrane Protein Oligomerization 253
9.2.7 Post–Translational Modifications 254
9.2.7.1 Glycosylation 255
9.2.7.2 Palmitoylation 255
9.2.8 Sequence Modifications 256
9.2.9 Lipids and Water 258
9.2.10 Purity and Contaminants 260
9.2.11 Current Trends in the Crystallization of a–Helical Membrane Proteins 260
9.3 Test Cases 261
9.3.1 Rhodopsin 261
9.3.2 RPE65 264
9.3.2.1 Expression in E. coli 264
9.3.2.2 Expression in Sf 9 Cells 264
9.3.2.3 Purification from Native Sources 264
9.3.3 Transmembrane Domain of M2 Protein from Influenza A Virus 265
Acknowledgments 267
Abbreviations 267
References 267
Part Three Emerging Methods and Approaches 275
10 Engineering Integral Membrane Proteins for Expression and Stability 277
I gor Dodevski and Andreas Plückthun
10.1 Introduction 277
10.2 Engineering Higher Expression 278
10.2.1 Directed Evolution of a GPCR for Higher Expression 280
10.2.2 Increasing Expression by Random Mutagenesis and Dot–Blot Based Screening 286
10.3 Engineering Higher Stability 288
10.3.1 Stabilizing a Prokaryotic IMP by Cysteine–Scanning, Random Mutagenesis, and Screening in a 96–Well Assay Format 289
10.3.2 Stabilizing GPCRs by Alanine–Scanning and Single–Clone Screening 290
10.3.3 Stabilizing GPCRs by Random Mutagenesis and Screening in a 96–Well Assay Format 291
10.4 Conclusions 294
Abbreviations 295
References 295
11 Expression and Purification of G–Protein–Coupled Receptors for Nuclear Magnetic Resonance Structural Studies 297
Fabio Casagrande, Klaus Maier, Hans Kiefer, Stanley J. Opella, and Sang Ho Park
11.1 Introduction: G–Protein–Coupled Receptor Superfamily 297
11.2 CXCR1 298
11.3 GPCR Structures 299
11.4 NMR Studies of GPCRs 300
11.5 Expression Systems 301
11.6 Cloning of CXCR1 into pGEX2a 303
11.7 Expression of CXCR1 304
11.8 Purification 305
11.9 Refolding and Reconstitution 306
11.10 Binding and Activity Measurements 307
11.10.1 NMR Samples 308
11.11 NMR Spectra 309
Acknowledgments 310
Abbreviations 311
References 311
12 Solubilization, Purification, and Characterization of Integral Membrane Proteins 317
Víctor Lórenz–Fonfría, Alex Perálvarez–Marín, Esteve Padrós, and Tzvetana Lazarova
12.1 Introduction 317
12.2 Solubilization of IMPs 319
12.2.1 Physicochemical Characteristics of Detergents 319
12.2.2 Classific