Membrane Protein Purification and Crystallization

A Practical Guide

By: Hermann Schagger

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Hardcover | 19 December 2002 | Edition Number 2

At a Glance

Hardcover
316 Pages

Edition Type
Revised

Dimensions(cm)
24.13 x 20.96 x 1.91

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This second edition of Membrane Protein Purification and Crystallization, A Practical Guide is written for bench scientists working in the fields of biochemistry, biology, and proteomic research. This guide presents isolation and crystallization techniques in a concise form, emphasizing the critical aspects unique to membrane proteins. It explains the principles of the methods and provides protocols of general use, permitting researchers and students new to this area to adapt these techniques to their particular needs. This edition is not only an update but is comprised mainly of new contributions. It is the first monograph compiling the essential approaches for membrane protein crystallization, and emphasizes recent progress in production and purification of recombinant membrane proteins.

Provides general guidelines and strategies for isolation and crystallization of membrane proteins
Gives detailed protocols that have wide application, and low specialized equipment needs
Emphasizes recent progress in production and purification of recombinant membrane proteins, especially of histidine-tagged and other affinity-epitope-tagged proteins
Summarizes recent developments of Blue-Native PAGE, a high resolution separation technique, which is independent of the use of recombinant techniques, and is especially suited for proteomic analyses of membrane protein complexes
Gives detailed protocols for membrane protein crystallization, and describes the production and use of antibody fragments for high resolution crystallization
Presents a comprehensive guide to 2D-crystallization of membrane proteins

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Contributors	p. xv
Preface	p. xvii
Strategies and Techniques
Purification Strategies for Membrane Proteins
Introduction	p. 1
General Guide for Retaining Catalytic Activity	p. 3
Choice and Sequence of Purification Techniques	p. 4
Choice of Protein Source, Disruption of Cells, and Preparation of Organelles and Membranes	p. 6
Protein Assay	p. 7
Catalytic Activity and Other Specific Properties	p. 7
Determination of Total Protein	p. 8
Solubilization and Stabilization of Membrane Proteins	p. 9
Choice of Buffer	p. 9
Choice of Detergent	p. 10
Protein Stability and Protease Inhibition	p. 16
References	p. 18
Techniques and Basic Operations in Membrane Protein Purification
Introduction	p. 19
Solubilization-Precipitation	p. 20
Differential Extraction	p. 20
Phase Separation and Differential Precipitation	p. 24
Centrifugation	p. 26
Concentration of Samples, Exchange of Buffer, and Exchange of Detergent	p. 26
Sample Concentration	p. 26
Exchange of Buffer	p. 28
Removal and Exchange of Detergent	p. 28
Chromatographic Techniques	p. 29
Hydroxylapatite Chromatography	p. 30
Ion-Exchange Chromatography	p. 35
Chromatofocusing	p. 38
Metal Chelate Chromatography	p. 43
Affinity and Dye-Ligand Chromatography	p. 45
Covalent Chromatography	p. 48
Hydrophobic Interaction Chromatography	p. 49
Gel Filtration	p. 49
References	p. 52
Production and Purification of Recombinant Membrane Proteins
Introduction	p. 55
Prokaryotic Expression Systems for Overproduction of Membrane Proteins for Structural Studies	p. 57
E. coli Inducible Promoters Used for Overexpression	p. 58
Optimizing Expression Levels in E. coli	p. 60
Inclusion Bodies and Refolding	p. 62
Eukaryotic Expression Systems for Overproduction of Membrane Proteins	p. 63
Yeast-Based Expression Systems	p. 64
Insect Cells as Expression System	p. 70
Use of Fusion Proteins and Affinity Tags for Purification of Membrane Proteins	p. 73
Acknowledgments	p. 77
References	p. 78
SDS Electrophoresis Techniques
Introduction	p. 85
Choice of Optimal SDS-Page System and Gel Type	p. 86
Wide versus Narrow Molecular Mass Range	p. 86
Separation of Proteins with Similar Molecular Masses	p. 86
Avoiding Broad Bands	p. 88
Analytical versus Preparative Purpose	p. 88
Tricine-SDS-PAGE	p. 88
Comparison with Laemmli System	p. 89
Characteristics of Gel Types	p. 89
Gel Preparation	p. 91
Sample Preparation and Protein Loading	p. 93
Electrophoresis Conditions	p. 95
Staining and Drying of High Percentage Polyacrylamide Gels	p. 96
Recovery of Proteins from SDS Gels	p. 97
Recovery of Proteins from Fixed and Coomassie-Stained SDS Gels	p. 97
Blue-SDS-PAGE	p. 97
Recovery of Membrane Proteins after Blue-SDS-PAGE	p. 99
Processing of Proteins Recovered from Gels for Immunization and Protein Sequencing	p. 100
Electroblotting	p. 101
References	p. 102
Blue Native Electrophoresis
Introduction	p. 105
Techniques	p. 106
Native First Dimension: BN-PAGE	p. 106
Native Second Dimension: BN-PAGE + Detergent	p. 116
Second or Third Dimension: SDS-PAGE	p. 116
Applications	p. 119
Final Purification of Crude Membrane Proteins for Immunization and Protein Sequencing	p. 120
Separation of Native Membrane Protein Complexes from Solubilized Biological Membranes	p. 120
References	p. 129
Preparative Isoelectric Focusing
Introduction	p. 131
Techniques	p. 133
Instruments and Accessories	p. 133
Analytical Electrofocusing	p. 134
Preparative Flat-Bed Electrofocusing	p. 136
Application: Purification of Photosystem I	p. 138
Conclusions	p. 139
References	p. 141
Membrane Protein Crystallization
Introduction	p. 143
Bottlenecks in Membrane Protein Crystallization	p. 146
Starting Material	p. 146
Protein Purification and Characterization	p. 147
Detergents	p. 151
Crystallization	p. 154
Conclusion	p. 156
References	p. 156
Discussion of Selected Isolation Protocols
Lipid-Dependent Inactivation and Reactivation of Bovine Complex III
Introduction	p. 161
Lipid-Dependent Inactivation and Reactivation	p. 162
Delipidation	p. 162
Reconstitution of Lipid-Dependent Catalytic Activity	p. 163
References	p. 165
Purification of an Affinity-Epitope Tagged G-Protein Coupled Receptor
Introduction	p. 167
Production of Recombinant [beta subscript 2]-Adrenergic Receptor in Pichia pastoris	p. 168
Media for Pichia pastoris	p. 168
Growth and Expression of Pichia pastoris Clones	p. 169
Preparation of Pichia pastoris Membranes	p. 169
Solubilization of Membranes	p. 170
Purification of the [beta subscript 2]-Adrenergic Receptor	p. 170
Ligand Affinity Chromatography of [beta subscript 2]-Adrenergic Receptor	p. 170
Anti-FLAG M1 Antibody Affinity Chromatography	p. 171
Affinity Chromatography of Biotinylated [beta subscript 2]-Adrenergic Receptor by Monomeric Avidin Resin (Optional)	p. 172
Analytical Gel Filtration (Optional)	p. 174
Ligand Binding Assays	p. 174
Binding Assay on Membranes	p. 174
Binding Assay for Solubilized Receptor	p. 175
References	p. 177
Purification of NhaA Na[superscript +]/H[superscript +] Antiporter of Escherichia Coli for 3D or 2D Crystallization
Introduction	p. 179
Growth of Escherichia coli for Production of His-Tagged NhaA in a 10 1 Fermenter	p. 180
Isolation of Membranes from Escherichia coli TA16/pAXH Strain	p. 182
Two-Step Purification of His-Tagged NHaA	p. 183
Affinity Purification of His-Tagged NhaA on the Ni[superscript 2+]-NTA Matrix	p. 183
Purification of His-Tagged NhaA by Gel Filtration Chromatography	p. 184
NhaA Reconstitution into Liposomes and Activity Assay	p. 185
Reconstitution by Detergent Dilution	p. 185
Reconstitution by BioBeads	p. 187
NhaA Activity Assay: [Delta]pH Driven [superscript 22]Na[superscript +] Active Transport	p. 187
Dynamic Light Scattering Experiments	p. 189
Acknowledgments	p. 189
References	p. 190
Purification of the Cytochrome bc[subscript 1] Complex from Yeast
Introduction	p. 191
Preparation of Membranes from Saccharomyces cerevisiae	p. 192
Cytochrome bc[subscript 1] Complex Preparation	p. 193
Detergent Solubilization of Membranes	p. 193
Purification of the Cytochrome bc[subscript 1] Complex by Anion-Exchange Chromatography	p. 194
Size Exclusion Chromatography	p. 196
Protein Determination	p. 196
Spectroscopic Quantification of the Cytochrome bc[subscript 1] Complex	p. 197
Determination of Cytochrome bc[subscript 1] Complex Activity	p. 197
Analytical Size Exclusion Chromatography	p. 198
SDS-PAGE Analysis	p. 198
Conclusions	p. 200
Acknowledgments	p. 201
References	p. 202
Crystallization of Membrane Proteins
Antibody Fragment Mediated Crystallization of Membrane Proteins
Introduction	p. 205
Production of Antibody Fv Fragments in the Periplasm of E. coli	p. 210
Purification of Fvs by Streptavidin Affinity Chromatography	p. 210
Fv Mediated Crystallization of Cytochrome c Oxidase from P. denitrificans	p. 211
Growth of P. denitrificans	p. 211
Isolation of Membranes	p. 211
Solubilization of Membrane Proteins	p. 212
Indirect Immunoaffinity Chromatography	p. 212
Detergent Exchange and Size Exclusion Chromatography	p. 212
Crystallization of Cytochrome c Oxidase-Fv Complex	p. 214
Fv-Mediated Crystallization of the Cytochrome bc[subscript 1] Complex from the Yeast S. cerevisiae	p. 214
Co-Complex Formation and Purification	p. 214
Crystallization of Cytochrome bc[subscript 1] Complex: Fv18E11 Co-Complex	p. 215
Conclusions	p. 217
Acknowledgments	p. 217
References	p. 217
Crystallization of Wolinella succinogenes Quinol: Fumarate Reductase
Introduction	p. 219
Preparatory Steps	p. 221
Growth of Wolinella succinogenes	p. 221
Isolation of Quinol: Fumarate Reductase	p. 222
Crystallization of Quinol: Fumarate Reductase	p. 225
Conclusions	p. 226
References	p. 227
Ba[subscript 3]-Type Cytochrome c Oxidase from Thermus thermophilus: Purification, Crystallization, and Crystal Transformation
Introduction	p. 229
Fermentation, Purification, and Molecular Characterization	p. 231
Fermentation	p. 231
Preparation Protocol	p. 231
Molecular Characterization	p. 234
Crystallization and Initial Crystallographic Characterization	p. 236
Detergent Exchange	p. 236
Crystallization in Sitting-Drops	p. 237
Crystallization in Batch	p. 238
Initial Crystallographic Characterization	p. 239
Crystal Transformation	p. 240
Oil Method	p. 240
Transformation and Freezing with the Humidity Machine	p. 243
Acknowledgments	p. 249
References	p. 249
Two-Dimensional Crystallization of Membrane Proteins: A Practical Guide
Introduction	p. 253
Things to Consider Before You Start	p. 254
2D or 3D Crystals?	p. 254
Which Membrane Protein Structures Have Been Determined by Electron Microscopy?	p. 254
Crystallization Parameters	p. 255
The Protein	p. 255
The Lipid	p. 260
The Detergent	p. 263
pH	p. 264
Salt Concentration and Ionic Additives	p. 265
Nonionic Additives	p. 265
Precipitants	p. 265
Temperature	p. 266
Methods for Growing 2D Crystals of Membrane Proteins	p. 267
2D Crystallization in Suspension	p. 267
2D Crystallization on a Surface	p. 273
2D Crystallization in situ	p. 276
How to Find 2D Crystals	p. 277
Outlook	p. 279
References	p. 280
In Cubo Crystallization of Membrane Proteins
Introduction	p. 285
Properties of Lipidic Cubic Phases	p. 287
Practical Aspects of Lipidic Cubic Phases	p. 290
Setting Up In Cubo Crystallization	p. 290
Establishing In Cubo Crystallization Conditions	p. 295
Harvesting Single Crystals from the Lipidic Cubic Phase Matrix	p. 296
Mechanism of Crystallization In Cubo	p. 298
Summary	p. 300
Acknowledgments	p. 301
References	p. 301
Index	p. 303
Table of Contents provided by Syndetics. All Rights Reserved.

Membrane Protein Purification and Crystallization

A Practical Guide

At a Glance

Hardcover

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How to return your order

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