Contributors

Preface

xvii

I STRATEGIES AND TECHNIQUES

Purification Strategies for Membrane Proteins

Gebhard Von Jagow

Thomas A. Link

Hermann Schagger

Introduction

(2)

General Guide for Retaining Catalytic Activity

(1)

Choice and Sequence of Purification Techniques

(2)

Choice of Protein Source, Disruption of Cells, and Preparation of Organelles and Membranes

(1)

Protein Assay

(2)

Catalytic Activity and Other Specific Properties

(1)

Determination of Total Protein

(1)

Solubilization and Stabilization of Membrane Proteins

(10)

Choice of Buffer

(1)

Choice of Detergent

(6)

Protein Stability and Protease Inhibition

(2)

References

(1)

Techniques and Basic Operations in Membrane Protein Purification

Hermann Schagger

Introduction

(1)

Solubilization-Precipitation

(6)

Differential Extraction

(4)

Phase Separation and Differential Precipitation

(2)

Centrifugation

(1)

Concentration of Samples, Exchange of Buffer, and Exchange of Detergent

(2)

Exchange of Detergent

(1)

Sample Concentration

(2)

Exchange of Buffer

(1)

Removal and Exchange of Detergent

(27)

Chromatographic Techniques

(1)

Hydroxylapatite Chromatography

(5)

Ion-Exchange Chromatography

(3)

Chromatofocusing

(5)

Metal Chelate Chromatography

(2)

Affinity and Dye-Ligand Chromatography

(3)

Covalent Chromatography

(1)

Hydrophobic Interaction Chromatography

(1)

Gel Filtration

(3)

References

(3)

Production and Purification of Recombinant Membrane Proteins

Etana Padan

Carola Hunte

Helmut Reilander

Introduction

(2)

Prokaryotic Expression Systems for Overproduction of Membrane Proteins for Structural Studies

(6)

E. coli Inducible Promoters Used for Overexpression

(2)

Optimizing Expression Levels in E. coli

(2)

Inclusion Bodies and Refolding

(1)

Eukaryotic Expression Systems for Overproduction of Membrane Proteins

(10)

Yeast-Based Expression Systems

(6)

Insect Cells as Expression System

(3)

Use of Fusion Proteins and Affinity Tags for Purification of Membrane Proteins

(12)

Acknowledgments

(1)

References

(7)

SDS Electrophoresis Techniques

Hermann Schagger

Introduction

(1)

Choice of Optimal SDS-Page System and Gel Type

(2)

Wide versus Narrow Molecular Mass Range

(1)

Separation of Proteins with Similar Molecular Masses

(2)

Avoiding Broad Bands

(1)

Analytical versus Preparative Purpose

(1)

Tricine-SDS-Page

(9)

Comparison with Laemmli System

(1)

Characteristics of Gel Types

(2)

Gel Preparation

(2)

Sample Preparation and Protein Loading

(2)

Electrophoresis Conditions

(1)

Staining and Drying of High Percentage Polyacrylamide Gels

(1)

Recovery of Proteins from SDS Gels

(3)

Recovery of Proteins from Fixed and Coomassie-Stained SDS Gels

(1)

Blue-SDS-Page

(2)

Recovery of Membrane Proteins after Blue-SDS-Page

(1)

Processing of Proteins Recovered from Gels for Immunization and Protein Sequencing

100

(1)

Electroblotting

101

(4)

References

102

(3)

Blue Native Electrophoresis

Hermann Schagger

Introduction

105

(1)

Techniques

106

(13)

Native First Dimension: BN-Page

106

(10)

Native Second Dimension: BN-Page + Detergent

116

(1)

Second or Third Dimension: SDS-Page

116

(3)

Applications

119

(12)

Final Purification of Crude Membrane Proteins for Immunization and Protein Sequencing

120

(1)

Separation of Native Membrane Protein Complexes from Solubilized Biological Membranes

120

(9)

References

129

(2)

Preparative Isoelectric Focusing

Irene Tsirogianni

Georgios Tsiotis

Introduction

131

(2)

Techniques

133

(5)

Instruments and Accessories

133

(1)

Analytical Electrofocusing

134

(2)

Preparative Flat-Bed Electrofocusing

136

(2)

Application: Purification of Photosystem I

138

(1)

Conclusions

139

(4)

References

141

(2)

Membrane Protein Crystallization

Carola Hunte

Hartmut Michel

Introduction

143

(3)

Bottlenecks in Membrane Protein Crystallization

146

(15)

Starting Material

146

(1)

Protein Purification and Characterization

147

(4)

Detergents

151

(3)

Crystallization

154

(2)

Conclusion

156

(1)

References

156

(5)

II DISCUSSION OF SELECTED ISOLATION PROTOCOLS

Lipid-Dependent Inactivation and Reactivation of Bovine Complex III

Hermann Schagger

Introduction

161

(1)

Lipid-Dependent Inactivation and Reactivation

162

(5)

Delipidation

162

(1)

Reconstitution of Lipid-Dependent Catalytic Activity

163

(2)

References

165

(2)

Purification of an Affinity-Epitope Tagged G-Protein Coupled Receptor

Christoph Reinhart

H Markus Weiss

Helmut Reilander

Introduction

167

(1)

Production fo Recombinant β2-Adrenergic Receptor in Pichia pastoris

168

(1)

Media for Pichia pastoris

168

(1)

Growth and Expression of Pichia pastoris Clones

169

(1)

Preparation of Pichia pastoris Membranes

169

(1)

Solubilization of Membranes

170

(1)

Purification of the β2-Adrenergic Receptor

170

(4)

Ligand Affinity Chromatography of β2-Adrenergic Receptor

170

(1)

Anti-Flag M1 Antibody Affinity Chromatography

171

(1)

Affinity Chromatography of Biotinylated β2-Adrenergic Receptor by Monomeric Avidin Resin (Optional)

172

(2)

Analytical Gel Filtration (Optional)

174

(1)

Ligand Binding Assays

174

(5)

Binding Assay on Membranes

174

(1)

Binding Assay for Solubilized Receptor

175

(2)

References

177

(2)

Purification of NhaA Na+/H+ Antiporter of Escherichia Coli for 3D or 2D Crystallization

Miro Venturi

Etana Padan

Introduction

179

(1)

Growth of Escherichia coli for Production of His-Tagged NhaA in a 10 1 Fermenter

180

(2)

Isolation of Membranes from Escherichia coli TA16/pAXH Strain

182

(1)

Two-Step Purification of His-Tagged NHaA

183

(2)

Affinity Purification of His-Tagged NhaA on the Ni2+-NTA Matrix

183

(1)

Purification of His-Tagged NhaA by Gel Filtration Chromatography

184

(1)

NhaA Reconstitution into Liposomes and Activity Assay

185

(4)

Reconstitution by Detergent Dilution

185

(2)

Reconstitution by BioBeads

187

(1)

NhaA Activity Assay: ΔpH Driven 22Na+ Active Transport

187

(2)

Dynamic Light Scattering Experiments

189

(2)

Acknowledgments

189

(1)

References

190

(1)

Purification of the Cytochrome bc1 Complex from Yeast

Hildur Palsdottir

Carola Hunte

Introduction

191

(1)

Preparation of Membranes from Saccharomyces cerevisiae

192

(1)

Cytochrome bc1 Complex Preparation

193

(7)

Detergent Solubilization of Membranes

193

(1)

Purification of the Cytochrome bc1 Complex by Anion-Exchange Chromatography

194

(2)

Size Exclusion Chromatography

196

(1)

Protein Determination

196

(1)

Spectroscopic Quantification of the Cytochrome bc1 Complex

197

(1)

Determination of Cytochrome bc1 Complex Activity

197

(1)

Analytical Size Exclusion Chromatography

198

(1)

SDS-Page Analysis

198

(2)

Conclusions

200

(5)

Acknowledgments

201

(1)

References

202

(3)

III CRYSTALLIZATION OF MEMBRANE PROTEINS

Antibody Fragment Mediated Crystallization of Membrane Proteins

Carola Hunte

Aimo Kannt

Introduction

205

(5)

Production of Antibody Fv Fragments in the Periplasm of E. coli

210

(1)

Purification of Fvs by Streptavidin Affinity Chromatography

210

(1)

Fv Mediated Crystallization of Cytochrome c Oxidase from P. denitrificans

211

(3)

Growth of P. denitrificans

211

(1)

Isolation of Membranes

211

(1)

Solubilization of Membrane Proteins

212

(1)

Indirect Immunoaffinity Chromatography

212

(1)

Detergent Exchange and Size Exclusion Chromatography

212

(2)

Crystallization of Cytochrome c Oxidase-Fv Complex

214

(1)

Fv-Mediated Crystallization of the Cytochrome bc1 Complex from the Yeast S. cerevisiae

214

(3)

Co-Complex Formation and Purification

214

(1)

Crystallization of Cytochrome bc1 Complex: Fv18E11 Co-Complex

215

(2)

Conclusions

217

(2)

Acknowledgments

217

(1)

References

217

(2)

Crystallization of Wolinella succinogenes Quinol: Fumarate Reductase

C. Roy D. Lancaster

Introduction

219

(2)

Preparatory Steps

221

(4)

Growth of Wolinella succinogenes

221

(1)

Isolation of Quinol: Fumarate Reductase

222

(3)

Crystallization of Quinol: Fumarate Reductase

225

(1)

Conclusions

226

(3)

References

227

(2)

Ba3-Type Cytochrome c Oxidase from Thermus thermophilus: Purification, Crystallization, and Crystal Transformation

Tewfik Soulimane

Reiner Kiefersauer

Manuel E. Than

Introduction

229

(2)

Fermentation, Purification, and Molecular Characterization

231

(5)

Fermentation

231

(1)

Preparation Protocol

231

(3)

Molecular Characterization

234

(2)

Crystallization and Initial Crystallographic Characterization

236

(4)

Detergent Exchange

236

(1)

Crystallization in Sitting-Drops

237

(1)

Crystallization in Batch

238

(1)

Initial Crystallographic Characterization

239

(1)

Crystal Transformation

240

(13)

Oil Method

240

(3)

Transformation and Freezing with the Humidity Machine

243

(6)

Acknowledgments

249

(1)

References

249

(4)

Two-Dimensional Crystallization of Membrane Proteins: A Practical Guide

Werner Kuhlbrandt

Introduction

253

(1)

Things to Consider Before You Start

254

(1)

2D or 3D Crystals?

254

(1)

Which Membrane Protein Structures Have Been Determined by Electron Microscopy?

254

(1)

Crystallization Parameters

255

(12)

The Protein

255

(5)

The Lipid

260

(3)

The Detergent

263

(1)

264

(1)

Salt Concentration and Ionic Additives

265

(1)

Nonionic Additives

265

(1)

Precipitants

265

(1)

Temperature

266

(1)

Methods for Growing 2D Crystals of Membrane Proteins

267

(10)

2D Crystallization in Suspension

267

(6)

2D Crystallization on a Surface

273

(3)

2D Crystallization in situ

276

(1)

How to Find 2D Crystals

277

(2)

Outlook

279

(6)

References

280

(5)

In Cubo Crystallization of Membrane Proteins

Ehud M. Landau

Introduction

285

(2)

Properties of Lipidic Cubic Phases

287

(3)

Practical Aspects of Lipidic Cubic Phases

290

(8)

Setting Up In Cubo Crystallization

290

(5)

Establishing In Cubo Crystallization Conditions

295

(1)

Harvesting Single Crystals from the Lipidic Cubic Phase Matrix

296

(2)

Mechanism of Crystallization In Cubo

298

(2)

Summary

300

(3)

Acknowledgments

301

(1)

References

301

(2)

Index

303

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Membrane Protein Purification and Crystallization

9780123617767

0123617766

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