Membrane protein purification and crystallization: a practical guide
Saved in:
| Format: | Book |
|---|---|
| Language: | English |
| Published: |
Amsterdam [u.a.]
Acad. Press
2003
|
| Edition: | 2. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XVIII, 316 S. Ill., graph. Darst. |
| ISBN: | 0123617766 |
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MARC
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| 245 | 1 | 0 | |a Membrane protein purification and crystallization |b a practical guide |c ed. by Carola Hunte ... |
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Record in the Search Index
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| adam_text | vi Contents
III. Choice and Sequence of Purification Techniques 4
IV. Choice of Protein Source, Disruption of Cells, and
Preparation of Organelles and Membranes 6
V. Protein Assay 7
A. Catalytic Activity and Other Specific Properties 7
B. Determination of Total Protein 8
VI. Solubilization and Stabilization of Membrane Proteins 9
A. Choice of Buffer 9
B. Choice of Detergent 10
C. Protein Stability and Protease Inhibition 16
References 18
O Techniques and Basic Operations in Membrane Protein
Purification
HERMANN SCHAGGER
I. Introduction 19
II. Solubilization Precipitation 20
A. Differential Extraction 20
B. Phase Separation and Differential Precipitation 24
C. Centrifugation 26
III. Concentration of Samples, Exchange of Buffer, and
Exchange of Detergent 26
A. Sample Concentration 26
B. Exchange of Buffer 28
C. Removal and Exchange of Detergent 28
IV. Chromatographic Techniques 29
A. Hydroxylapatite Chromatography 30
B. Ion Exchange Chromatography 35
C. Chromatofocusing 38
D. Metal Chelate Chromatography 43
E. Affinity and Dye Ligand Chromatography 45
F. Covalent Chromatography 48
G. Hydrophobic Interaction Chromatography 49
H. Gel Filtration 49
References 52
Q Production and Purification of Recombinant Membrane
Proteins
ETANA PADAN, CAROLA HUNTE, AND HELMUT REILANDER
I. Introduction 55
Contents vii
II. Prokaryotic Expression Systems for Overproduction of
Membrane Proteins for Structural Studies 57
A. E. coli Inducible Promoters Used for Overexpression 58
B. Optimizing Expression Levels in E. coli 60
C. Inclusion Bodies and Refolding 62
III. Eukaryotic Expression Systems for Overproduction of
Membrane Proteins 63
A. Yeast Based Expression Systems 64
B. Insect Cells as Expression System 70
IV. Use of Fusion Proteins and Affinity Tags for Purification
of Membrane Proteins 73
Acknowledgments 77
References 78
A SDS Electrophoresis Techniques
HERMANN SCHAGGER
I. Introduction 85
II. Choice of Optimal SDS Page System and Gel Type 86
A. Wide versus Narrow Molecular Mass Range 86
B. Separation of Proteins with Similar Molecular Masses 86
C. Avoiding Broad Bands 88
D. Analytical versus Preparative Purpose 88
III. Tricine SDS PAGE 88
A. Comparison with Laemmli System 89
B. Characteristics of Gel Types 89
C. Gel Preparation 91
D. Sample Preparation and Protein Loading 93
E. Electrophoresis Conditions 95
F. Staining and Drying of High Percentage Polyacrylamide Gels 96
IV. Recovery of Proteins from SDS Gels 97
A. Recovery of Proteins from Fixed and Coomassie Stained SDS Gels 97
B. Blue SDS PAGE 97
C. Recovery of Membrane Proteins after Blue SDS PAGE 99
V. Processing of Proteins Recovered from Gels for Immunization and Protein
Sequencing 100
VI. Electroblotting 101
References 102
C Blue Native Electrophoresis
HERMANN SCHAGGER
I. Introduction 105
viii Contents
II. Techniques 106
A. Native First Dimension: BN PAGE 106
B. Native Second Dimension: BN PAGE + Detergent 116
C. Second or Third Dimension: SDS PAGE 116
III. Applications 119
A. Final Purification of Crude Membrane Proteins for Immunization
and Protein Sequencing 120
B. Separation of Native Membrane Protein Complexes from Solubilized
Biological Membranes 120
References 129
C* Preparative Isoelectric Focusing
IRENE TSIROGIANNI AND GEORGIOS TSIOTIS
I. Introduction 131
II. Techniques 133
A. Instruments and Accessories 133
B. Analytical Electrofocusing 134
C. Preparative Flat Bed Electrofocusing 136
III. Application: Purification of Photosystem I 138
IV. Conclusions 139
References 141
7 Membrane Protein Crystallization
CAROLA HUNTE AND HARTMUT MICHEL
I. Introduction 143
II. Bottlenecks in Membrane Protein Crystallization 146
A. Starting Material 146
B. Protein Purification and Characterization 147
C. Detergents 151
D. Crystallization 154
E. Conclusion 156
References 156
Contents IX
11 DISCUSSION OF SELECTED ISOLATION PROTOCOLS
Q Lipid Dependent Inactivation and Reactivation
° of Bovine Complex III
HERMANN SCHAGGER
I. Introduction 161
II. Lipid Dependent Inactivation and Reactivation 162
A. Delipidation 162
B. Reconstitution of Lipid Dependent Catalytic Activity 163
References 165
Q Purification of an Affinity Epitope Tagged G Protein
Coupled Receptor
CHRISTOPH REINHART, H MARKUS WEISS, AND HELMUT REILANDER
I. Introduction 167
II. Production of Recombinant /?2 Adrenergic Receptor in Pichia pastoris 168
A. Media for Pichia pastoris 168
B. Growth and Expression of Pichia pastoris Clones 169
III. Preparation of Pichia pastoris Membranes 169
IV. Solubilization of Membranes 170
V. Purification of the /J2 Adrenergic Receptor 170
A. Ligand Affinity Chromatography of /)2 Adrenergic Receptor 170
B. Anti FLAG Ml Antibody Affinity Chromatography 171
C. Affinity Chromatography of Biotinylated /?2 Adrenergic Receptor by
Monomeric Avidin Resin (Optional) 172
D. Analytical Gel Filtration (Optional) 174
VI. Ligand Binding Assays 174
A. Binding Assay on Membranes 174
B. Binding Assay for Solubilized Receptor 175
References 177
I C Purification of NhaA Na+/H+ Antiporter of Escherichia Coli
1 ^ for 3D or 2D Crystallization
MIRO VENTURI AND ETANA PAD AN
I. Introduction 179
II. Growth of Escherichia coli for Production of His Tagged NhaA in a 10 1
Fermenter 180
X Contents
III. Isolation of Membranes from Escherichia coli TA16/pAXH Strain 182
IV. Two Step Purification of His Tagged NHaA 183
A. Affinity Purification of His Tagged NhaA on the Ni2+ NTA Matrix 183
B. Purification of His Tagged NhaA by Gel Filtration Chromatography 184
V. NhaA Reconstitution into Liposomes and Activity Assay 185
A. Reconstitution by Detergent Dilution 185
B. Reconstitution by BioBeads 187
C. NhaA Activity Assay: ApH Driven 22Na+ Active Transport 187
VI. Dynamic Light Scattering Experiments 189
Acknowledgments 189
References 190
1 I Purification of the Cytochrome bc Complex from Yeast
HILDUR PALSDOTTIR AND CAROLA HUNTE
I. Introduction 191
II. Preparation of Membranes from Saccharomyces cerevisiae 192
III. Cytochrome bc1 Complex Preparation 193
A. Detergent Solubilization of Membranes 193
B. Purification of the Cytochrome bc1 Complex by Anion Exchange Chromato¬
graphy 194
C. Size Exclusion Chromatography 196
D. Protein Determination 196
E. Spectroscopic Quantification of the Cytochrome bct Complex 197
F. Determination of Cytochrome bc1 Complex Activity 197
G. Analytical Size Exclusion Chromatography 198
H. SDS PAGE Analysis 198
IV. Conclusions 200
Acknowledgments 201
References 202
111 CRYSTALLIZATION OF MEMBRANE PROTEINS
1O Antibody Fragment Mediated Crystallization of
Membrane Proteins
CAROLA HUNTE AND AIMO KANNT
I. Introduction 205
II. Production of Antibody Fv Fragments in the Periplasm of E. coli 210
Contents xi
III. Purification of Fvs by Streptavidin Affinity Chromatography 210
IV. Fv Mediated Crystallization of Cytochrome c Oxidase from
P. denitrificans 211
A. Growth of P. denitrificans 211
B. Isolation of Membranes 211
C. Solubilization of Membrane Proteins 212
D. Indirect Immunoaffinity Chromatography 212
E. Detergent Exchange and Size Exclusion Chromatography 212
F. Crystallization of Cytochrome c Oxidase Fu Complex 214
V. Fu Mediated Crystallization of the Cytochrome bc1 Complex from the
Yeast S. cerevisiae 214
A. Co Complex Formation and Purification 214
B. Crystallization of Cytochrome bct Complex: Fcl8Ell Co Complex 215
VI. Conclusions 217
Acknowledgments 217
References 217
I Q Crystallization of Wolinella succinogenes Quinol: Fumarate
Reductase
C. ROY D. LANCASTER
I. Introduction 219
II. Preparatory Steps 221
A. Growth of Wolinella succinogenes 221
B. Isolation of Quinol: Fumarate Reductase 222
III. Crystallization of Quinol: Fumarate Reductase 225
IV. Conclusions 226
References 227
I A. Bfl3 Type Cytochrome c Oxidase from Thermus thermophilus:
Purification, Crystallization, and Crystal Transformation
TEWFIK SOULIMANE, REINER KIEFERSAUER, AND
MANUEL E. THAN
I. Introduction 229
II. Fermentation, Purification, and Molecular Characterization 231
A. Fermentation 231
B. Preparation Protocol 231
C. Molecular Characterization 234
III. Crystallization and Initial Crystallographic Characterization 236
xii Contents
A. Detergent Exchange 236
B. Crystallization in Sitting Drops 237
C. Crystallization in Batch 238
D. Initial Crystallographic Characterization 239
IV. Crystal Transformation 240
A. Oil Method 240
B. Transformation and Freezing with the Humidity Machine 243
Acknowledgments 249
References 249
1 R Two Dimensional Crystallization of Membrane Proteins:
A^ A Practical Guide
WERNER KUHLBRANDT
I. Introduction 253
II. Things to Consider Before You Start 254
A. 2D or 3D Crystals? 254
B. Which Membrane Protein Structures Have Been Determined by Electron
Microscopy? 254
III. Crystallization Parameters 255
A. The Protein 255
B. TheLipid 260
C. The Detergent 263
D. pH 264
E. Salt Concentration and Ionic Additives 265
F. Nonionic Additives 265
G. Precipitants 265
H. Temperature 266
IV. Methods for Growing 2D Crystals of Membrane Proteins 267
A. 2D Crystallization in Suspension 267
B. 2D Crystallization on a Surface 273
C. 2D Crystallization in situ 276
V. How to Find 2D Crystals 277
VI. Outlook 279
References 280
1 C* In Cubo Crystallization of Membrane Proteins
EHUD M. LANDAU
I. Introduction 285
II. Properties of Lipidic Cubic Phases 287
Contents xiii
III. Practical Aspects of Lipidic Cubic Phases 290
A. Setting Up In Cubo Crystallization 290
B. Establishing In Cubo Crystallization Conditions 295
C. Harvesting Single Crystals from the Lipidic Cubic Phase Matrix 296
IV. Mechanism of Crystallization In Cubo 298
V. Summary 300
Acknowledgments 301
References 301
Index 303
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| indexdate | 2024-07-09T19:13:08Z |
| institution | BVB |
| isbn | 0123617766 |
| language | English |
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| physical | XVIII, 316 S. Ill., graph. Darst. |
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| publisher | Acad. Press |
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| spelling | Membrane protein purification and crystallization a practical guide ed. by Carola Hunte ... 2. ed. Amsterdam [u.a.] Acad. Press 2003 XVIII, 316 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Kristallisation - Membranproteine Membranproteine - Reinigungsverfahren Membranproteine (DE-588)4130026-9 gnd rswk-swf Kristallisation (DE-588)4033215-9 gnd rswk-swf Reinigungsverfahren (DE-588)4391913-3 gnd rswk-swf Membranproteine (DE-588)4130026-9 s Reinigungsverfahren (DE-588)4391913-3 s DE-604 Kristallisation (DE-588)4033215-9 s Hunte, Carola Sonstige oth HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010286338&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Membrane protein purification and crystallization a practical guide Kristallisation - Membranproteine Membranproteine - Reinigungsverfahren Membranproteine (DE-588)4130026-9 gnd Kristallisation (DE-588)4033215-9 gnd Reinigungsverfahren (DE-588)4391913-3 gnd |
| subject_GND | (DE-588)4130026-9 (DE-588)4033215-9 (DE-588)4391913-3 |
| title | Membrane protein purification and crystallization a practical guide |
| title_auth | Membrane protein purification and crystallization a practical guide |
| title_exact_search | Membrane protein purification and crystallization a practical guide |
| title_full | Membrane protein purification and crystallization a practical guide ed. by Carola Hunte ... |
| title_fullStr | Membrane protein purification and crystallization a practical guide ed. by Carola Hunte ... |
| title_full_unstemmed | Membrane protein purification and crystallization a practical guide ed. by Carola Hunte ... |
| title_short | Membrane protein purification and crystallization |
| title_sort | membrane protein purification and crystallization a practical guide |
| title_sub | a practical guide |
| topic | Kristallisation - Membranproteine Membranproteine - Reinigungsverfahren Membranproteine (DE-588)4130026-9 gnd Kristallisation (DE-588)4033215-9 gnd Reinigungsverfahren (DE-588)4391913-3 gnd |
| topic_facet | Kristallisation - Membranproteine Membranproteine - Reinigungsverfahren Membranproteine Kristallisation Reinigungsverfahren |
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