Guide to Protein Purification (eBook)

Front Cover 1
Methods in Enzymology: Guide to Protein Purification, 2nd Edition 4
Copyright Page 5
Contents 6
Contributors 20
Preface 26
Methods in Enzymology 28
Preface to Chapter 1 56
Chapter 1: Why Purify Enzymes? 58
Acknowledgment 61
Section 1: Developing Purification Procedures 62
Chapter 2: Strategies and Considerations for Protein Purifications 64
1. General Considerations 65
2. Source of the Protein 70
3. Preparing Extracts 71
4. Bulk or Batch Procedures for Purification 72
5. Refined Procedures for Purification 73
6. Conclusions 74
References 74
Chapter 3: Use of Bioinformatics in Planning a Protein Purification 76
1. What You Can Learn from an Amino Acid Sequence 77
2. What You Cannot yet Predict 80
3. Conclusion 81
References 82
Chapter 4: Preparing a Purification Summary Table 84
1. Introduction 84
2. The Importance of Footnotes 87
3. The Value of an SDS-Polyacrylamide Gel Analysis on Main Protein Fractions 87
4. Some Common Mistakes and Problems 87
Section 2: General Methods for Handling Proteins and Enzymes 90
Chapter 5: Setting Up a Laboratory 92
1. Supporting Materials 93
2. Detection and Assay Requirements 94
3. Fractionation Requirements 95
Chapter 6: Buffers: Principles and Practice 98
1. Introduction 98
2. Theory 99
3. Buffer Selection 100
4. Buffer Preparation 103
5. Volatile Buffers 104
6. Broad-Range Buffers 105
7. Recipes for Buffer Stock Solutions 105
References 111
Chapter 7: Measurement of Enzyme Activity 112
1. Introduction 113
2. Principles of Catalytic Activity 113
3. Measurement of Enzyme Activity 119
4. Formulation of Reaction Assay Mixtures 124
5. Discussion 126
Acknowledgments 126
References 126
Chapter 8: Quantitation of Protein 128
1. Introduction 129
2. General Instructions for Reagent Preparation 130
3. Ultraviolet Absorption Spectroscopy 135
4. Dye-Based Protein Assays 138
5. Coomassie Blue (Bradford) Protein Assay (Range: 1-50 mug) 140
6. Lowry (Alkaline Copper Reduction Assays) (Range: 5-100 mug) 141
7. Bicinchoninic Acid (BCA) (Range: 0.2-50 mug) 143
8. Amine Derivatization (Range: 0.05-25 mug) 144
9. Detergent-Based Fluorescent Detection (Range: 0.02-2 mug) 146
10. General Instructions 146
Acknowledgment 149
References 149
Chapter 9: Concentration of Proteins and Removal of Solutes 152
1. Chromatography 153
2. Electrophoresis 158
3. Dialysis 159
4. Ultrafiltration 162
5. Lyophilization 168
6. Precipitation 171
7. Crystallization 173
References 173
Chapter 10: Maintaining Protein Stability 176
1. Causes of Protein Inactivation 176
2. General Handling Procedures 177
3. Concentration and Solvent Conditions 177
4. Stability Trials and Storage Conditions 178
5. Proteolysis and Protease Inhibitors 179
6. Loss of Activity 180
Section 3: Recombinant Protein Expression and Purification 184
Chapter 11: Selecting an Appropriate Method for Expressing a Recombinant Protein 186
1. Introduction 187
2. Escherichia coli 188
3. Pichia pastoris 190
4. Baculovirus/Insect Cells 191
5. Mammalian Cells 193
6. Protein Characteristics 194
7. Recombinant Protein Applications 198
8. Conclusion 199
References 199
Chapter 12: Bacterial Systems for Production of Heterologous Proteins 204
1. Introduction 205
2. Heterologous Protein Production Using Escherichia coli 205
3. Planning a Bacterial Expression Project 206
4. Evaluation of Project Requirements 207
5. Target Analysis 207
6. Cloning 208
7. Preparation of T4 DNA Polymerase-Treated DNA Fragments 210
8. Expression in the E. coli Cytoplasm 211
9. Expression of Cytoplasmic Targets in E. coli 212
10. Analysis of Heterologous Protein Expression in E. coli 212
11. Small-Scale Expression Cultures in Autoinduction Media Protocol 215
12. Periplasmic Expression of Proteins 215
13. Expression of Periplasmic Targets in E. coli 216
14. Small-Scale Osmotic Shock Protocol 217
15. Alternative Bacterial Systems for Heterologous Protein Production 219
16. Alternative Vector and Induction Conditions 220
17. Production Scale 221
Acknowledgment 221
References 221
Chapter 13: Expression in the Yeast Pichia pastoris 224
1. Introduction 225
2. Other Fungal Expression Systems 225
3. Culture Media and Microbial Manipulation Techniques 226
4. Genetic Strain Construction 227
5. Gene Preparation and Vector Selection 229
6. Transformation by Electroporation 231
7. DNA Preparation 231
8. Examination of Strains for Recombinant Protein Production 233
9. Assay Development-The Yeastern Blot 237
10. Posttranslational Modification of the Recombinant Protein (Proteinases and Glycosylation) 239
11. Selection for Multiple Copies of an Expression Cassette 240
References 242
Chapter 14: Baculovirus-Insect Cell Expression Systems 246
1. Introduction 247
2. A Brief Overview of Baculovirus Biology and Molecular Biology 248
3. Baculovirus Expression Vectors 250
4. Baculovirus Expression Vector Technology-The Early Years 251
5. Baculovirus Expression Vector Technology-Improved 253
6. Baculovirus Transfer Plasmid Modifications 253
7. Parental Baculovirus Genome Modifications 255
8. The Other Half of the Baculovirus-Insect Cell System 265
9. A New Generation of Insect Cell Hosts for Baculovirus Expression Vectors 267
10. Basic Baculovirus Protocols 269
References 273
Chapter 15: Recombinant Protein Production by Transient Gene Transfer into Mammalian Cells 278
1. Introduction 279
2. HEK293 and CHO Cell Lines Commonly Used in TGE Approaches 279
3. Expression Vectors for HEK293 and CHO Cells 281
4. Cultivation of HEK293 Cells and CHO Cell Lines in Suspension 283
5. Transfection Methods 283
6. Conclusions 289
Acknowledgments 289
References 290
Chapter 16: Tagging for Protein Expression 294
1. Introduction 295
2. Some Considerations When Designing a Tagged Protein 296
3. Protein Affinity Tags 300
4. Solubility Tags 304
5. Removal of Tags 306
6. Conclusions 308
Acknowledgment 309
References 309
Chapter 17: Refolding Solubilized Inclusion Body Proteins 314
1. Introduction 315
2. General Refolding Consideration 317
3. General Procedures 317
4. General Protocol 318
5. Comments on this General Procedure 319
6. Performing a Protein Refolding Test Screen 326
7. Other Refolding Procedures 330
8. Refolding Database: Refold 332
9. Strategies to Increase Proportion of Soluble Protein 332
10. Conclusion 334
References 334
Section 4: Preparation of extracts and subcellular fractionation 338
Chapter 18: Advances in Preparation of Biological Extracts for Protein Purification 340
1. Introduction 341
2. Chemical and Enzymatic Cell Disruption 342
3. Mechanical Cell Disruption 345
4. Concluding Remarks 348
5. Procedures, Reagents, and Tips for Cell Disruption 348
References 356
Chapter 19: Isolation of Subcellular Organelles and Structures 360
1. Introduction 361
2. Extraction and Prefractionation of Subproteomes 363
References 382
Section 5: Purification Procedures: Bulk Methods 384
Chapter 20: Protein Precipitation Techniques 386
1. Introduction 387
2. Ammonium Sulfate Precipitation 387
3. Polyethyleneimine Precipitation 392
4. Other Methods 396
5. General Procedures When Fractionating Proteins by Precipitation 396
References 397
Chapter 21: Affi-Gel Blue for Nucleic Acid Removal and Early Enrichment of Nucleotide Binding Proteins 398
1. A Representative Protocol 399
References 400
Section 6: Purification Procedures: Chromatographic Methods 402
Chapter 22: Ion-Exchange Chromatography 404
1. Introduction 404
2. Principle 406
3. Stationary Phases 408
4. Binding Conditions 410
5. Elution Conditions 416
6. Operation of Ion-Exchange Columns 418
7. Example: Separation of Complex Protein Mixture 421
8. Example: High-Resolution Separation with a Monolithic Column 422
References 425
Chapter 23: Gel Filtration 428
1. Principle 428
2. Practice 429
Chapter 24: Protein Chromatography on Hydroxyapatite Columns 442
1. Introduction 443
2. Mechanisms 444
3. Chemical Characteristics 447
4. Purification Protocol Development 451
5. Packing Laboratory-Scale Columns 452
6. Process-Scale Column Packing 454
7. Applications 456
References 457
Chapter 25: Theory and Use of Hydrophobic Interaction Chromatography in Protein Purification Applications 460
1. Theory 461
2. Latest Technology in HIC Adsorbents 463
3. Procedures for Use of HIC Adsorbents 464
References 468
Section 7: Purification Procedures: Affinity Methods 470
Chapter 26: Affinity Chromatography: General Methods 472
1. Introduction 473
2. Selection of Affinity Matrix 474
3. Selection of Ligands 478
4. Attachment Chemistry 484
5. Purification Method 488
References 490
Chapter 27: Immobilized-Metal Affinity Chromatography (IMAC): A Review 494
1. Overview on IMAC Ligands and Immobilized Ions 495
2. IMAC Applications 499
3. Conclusions 522
Acknowledgments 523
References 523
Chapter 28: Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography 530
1. Introduction 531
2. Polyol-Responsive Monoclonal Antibodies 532
3. Conclusions 547
Disclosure 548
References 548
Section 8: Purification Procedures: Electrophoretic Methods 550
Chapter 29: One-Dimensional Gel Electrophoresis 552
1. Background 553
2. Polyacrylamide Gels 555
3. Principle of Method 556
4. Procedure 557
5. Detection of Proteins in Gels 563
6. Marker Proteins 565
7. Molecular Weight Determination 566
8. Preparative Electrophoresis 566
References 568
Chapter 30: Isoelectric Focusing and Two-Dimensional Gel Electrophoresis 570
1. Introduction 571
2. Materials 582
3. Methods 583
References 593
Chapter 31: Protein Gel Staining Methods: An Introduction and Overview 596
1. Introduction 597
2. General Considerations 598
3. Instrumentation: Detection and Documentation 600
4. Total Protein Detection 600
5. Phosphoprotein Detection 611
6. Glycoprotein Detection 612
References 614
Chapter 32: Elution of Proteins from Gels 620
1. Introduction 620
2. Elution of Proteins from Gels by Diffusion 621
3. Replacing the SDS Gel with a Reverse Phase HPLC 625
4. Electrophoretic Elution 625
5. Conclusion 626
References 626
Chapter 33: Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection 628
1. Western Blotting 629
2. Types of Western Blots 630
3. Detection Methods 634
4. The Chemiluminescence Signal 638
5. Common Problems and their Explanations 643
6. Blotting and Optimization Protocols using Chemiluminescent Substrates 648
References 653
Section 9: Purification Procedures: Membrane Proteins And Glycoproteins 656
Chapter 34: Detergents: An Overview 658
1. Introduction 659
2. Detergent Structure 659
3. Properties of Detergents in Solution 660
4. Exploiting the Physicochemical Parameters of Detergents for Membrane Protein Purification 667
5. Detergent Removal and Detergent Exchange 668
6. Choosing the Right Detergent 668
7. Conclusions 670
Acknowledgments 671
References 671
Chapter 35: Purification of Membrane Proteins 674
1. Introduction 674
2. Preparation of Membranes 675
3. Solubilization of Native Membrane Proteins 677
4. Purification of Membrane Proteins 680
5. Detergent Removal and Detergent Exchange 683
6. Expression and Purification of Recombinant Integral Membrane Proteins 683
References 684
Chapter 36: Purification of Recombinant G-Protein-Coupled Receptors 686
1. Introduction 687
2. Solubilization: General Considerations 688
3. Purification: General Considerations 689
4. Solubilization and Purification of a Recombinant Neurotensin Receptor NTS1 692
5. Analysis of Purified NTS1 696
6. Conclusions 697
Acknowledgments 697
References 697
Chapter 37: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes 702
1. Introduction 703
2. Overview of Cell-Free Translation 705
3. Expression Vectors 706
4. Gene Cloning 707
5. PCR Product Cleanup 710
6. Flexi Vector and PCR Product Digestion Reaction 711
7. Ligation Reaction 712
8. Transformation Reaction 713
9. Purification of Plasmid DNA 714
10. Preparation of mRNA 715
11. Preparation of Liposomes 716
12. Wheat Germ Translation Reaction 717
13. Purification by Density Gradient Ultracentrifugation 720
14. Characterization of Proteoliposomes 722
15. Considerations for Scale-Up 724
16. Isotopic Labeling for Structural Studies 724
17. Conclusions 725
Acknowledgments 725
References 725
Section 10: Characterization of Purified Proteins 730
Chapter 38: Determination of Protein Purity 732
1. Composition-Based and Activity-Based Analyses 735
2. Electrophoretic Methods 736
3. Chromatographic Methods 740
4. Sedimentation Velocity Methods 742
5. Mass Spectrometry Methods 742
6. Light Scattering Methods 743
References 744
Chapter 39: Determination of Size, Molecular Weight, and Presence of Subunits 746
1. Introduction 747
2. Chemical Methods 750
3. Transport Methods 753
4. Scattering Methods 771
5. Presence of Subunits 774
References 776
Chapter 40: Identification and Quantification of Protein Posttranslational Modifications 780
1. Introduction 781
2. Enrichment Techniques for Identifying PTMs 786
3. Nitrosative Protein Modifications 795
4. Methylation and Acetylation 796
5. Mass Spectrometry Analysis 799
6. CID versus ECD versus ETD 802
7. Quantifying PTMs 805
8. Future Directions 811
Acknowledgments 813
References 813
Section 11: Additional Techniques 820
Chapter 41: Parallel Methods for Expression and Purification 822
1. Introduction 822
2. Strategies Based on End-Use 823
3. Parallel Cloning Strategies for Creating Expression Constructs 826
4. Small-Scale Expression Screening to Identify Suitable Constructs 829
5. Analytical Testing of Proteins for Selection 833
6. Large-Scale Parallel Expression 835
7. Conclusion 838
Acknowledgments 838
References 839
Chapter 42: Techniques to Isolate O2-Sensitive Proteins: [4Fe–4S]-FNR as an Example 842
1. Introduction 843
2. Anaerobic Isolation of 4Fe-FNR 845
3. Characterization of [4Fe-4S]2+ Cluster Containing FNR 854
4. Summary 858
References 858
Section 12: Concluding Remarks 862
Chapter 43: Rethinking Your Purification Procedure 864
1. Introduction 864
Chapter 44: Important but Little Known (or Forgotten) Artifacts in Protein Biochemistry 868
1. Introduction 869
2. SDS Gel Electrophoresis Sample Preparation 869
3. Buffers 871
4. Chromatography 872
5. Protein Absorption During Filtration 873
6. Chemical Leaching from Plasticware 874
7. Cyanate in Urea 874
References 875
Author Index 876
Subject Index 890
Color Plates 908