Guide to Protein Purification

Front Cover

Methods in Enzymology: Guide to Protein Purification, 2nd Edition

Copyright Page

Contents

Contributors

Preface

Methods in Enzymology

Preface to Chapter 1

Chapter 1: Why Purify Enzymes?

Acknowledgment

Section 1: Developing Purification Procedures

Chapter 2: Strategies and Considerations for Protein Purifications

1. General Considerations

2. Source of the Protein

3. Preparing Extracts

4. Bulk or Batch Procedures for Purification

5. Refined Procedures for Purification

6. Conclusions

References

Chapter 3: Use of Bioinformatics in Planning a Protein Purification

1. What You Can Learn from an Amino Acid Sequence

2. What You Cannot yet Predict

3. Conclusion

References

Chapter 4: Preparing a Purification Summary Table

1. Introduction

2. The Importance of Footnotes

3. The Value of an SDS-Polyacrylamide Gel Analysis on Main Protein Fractions

4. Some Common Mistakes and Problems

Section 2: General Methods for Handling Proteins and Enzymes

Chapter 5: Setting Up a Laboratory

1. Supporting Materials

2. Detection and Assay Requirements

3. Fractionation Requirements

Chapter 6: Buffers: Principles and Practice

1. Introduction

2. Theory

3. Buffer Selection

4. Buffer Preparation

5. Volatile Buffers

6. Broad-Range Buffers

7. Recipes for Buffer Stock Solutions

References

Chapter 7: Measurement of Enzyme Activity

1. Introduction

2. Principles of Catalytic Activity

3. Measurement of Enzyme Activity

4. Formulation of Reaction Assay Mixtures

5. Discussion

Acknowledgments

References

Chapter 8: Quantitation of Protein

1. Introduction

2. General Instructions for Reagent Preparation

3. Ultraviolet Absorption Spectroscopy

4. Dye-Based Protein Assays

5. Coomassie Blue (Bradford) Protein Assay (Range: 1-50 mug)

6. Lowry (Alkaline Copper Reduction Assays) (Range: 5-100 mug)

7. Bicinchoninic Acid (BCA) (Range: 0.2-50 mug)

8. Amine Derivatization (Range: 0.05-25 mug)

9. Detergent-Based Fluorescent Detection (Range: 0.02-2 mug)

10. General Instructions

Acknowledgment

References

Chapter 9: Concentration of Proteins and Removal of Solutes

1. Chromatography

2. Electrophoresis

3. Dialysis

4. Ultrafiltration

5. Lyophilization

6. Precipitation

7. Crystallization

References

Chapter 10: Maintaining Protein Stability

1. Causes of Protein Inactivation

2. General Handling Procedures

3. Concentration and Solvent Conditions

4. Stability Trials and Storage Conditions

5. Proteolysis and Protease Inhibitors

6. Loss of Activity

Section 3: Recombinant Protein Expression and Purification

Chapter 11: Selecting an Appropriate Method for Expressing a Recombinant Protein

1. Introduction

2. Escherichia coli

3. Pichia pastoris

4. Baculovirus/Insect Cells

5. Mammalian Cells

6. Protein Characteristics

7. Recombinant Protein Applications

8. Conclusion

References

Chapter 12: Bacterial Systems for Production of Heterologous Proteins

1. Introduction

2. Heterologous Protein Production Using Escherichia coli

3. Planning a Bacterial Expression Project

4. Evaluation of Project Requirements

5. Target Analysis

6. Cloning

7. Preparation of T4 DNA Polymerase-Treated DNA Fragments

8. Expression in the E. coli Cytoplasm

9. Expression of Cytoplasmic Targets in E. coli

10. Analysis of Heterologous Protein Expression in E. coli

11. Small-Scale Expression Cultures in Autoinduction Media Protocol

12. Periplasmic Expression of Proteins

13. Expression of Periplasmic Targets in E. coli

14. Small-Scale Osmotic Shock Protocol

15. Alternative Bacterial Systems for Heterologous Protein Production

16. Alternative Vector and Induction Conditions

17. Production Scale

Acknowledgment

References

Chapter 13: Expression in the Yeast Pichia pastoris

1. Introduction

2. Other Fungal Expression Systems

3. Culture Media and Microbial Manipulation Techniques

4. Genetic Strain Construction

5. Gene Preparation and Vector Selection

6. Transformation by Electroporation

7. DNA Preparation

8. Examination of Strains for Recombinant Protein Production

9. Assay Development-The Yeastern Blot

10. Posttranslational Modification of the Recombinant Protein (Proteinases and Glycosylation)

11. Selection for Multiple Copies of an Expression Cassette

References

Chapter 14: Baculovirus-Insect Cell Expression Systems

1. Introduction

2. A Brief Overview of Baculovirus Biology and Molecular Biology

3. Baculovirus Expression Vectors

4. Baculovirus Expression Vector Technology-The Early Years

5. Baculovirus Expression Vector Technology-Improved

6. Baculovirus Transfer Plasmid Modifications

7. Parental Baculovirus Genome Modifications

8. The Other Half of the Baculovirus-Insect Cell System

9. A New Generation of Insect Cell Hosts for Baculovirus Expression Vectors

10. Basic Baculovirus Protocols

References

Chapter 15: Recombinant Protein Production by Transient Gene Transfer into Mammalian Cells

1. Introduction

2. HEK293 and CHO Cell Lines Commonly Used in TGE Approaches

3. Expression Vectors for HEK293 and CHO Cells

4. Cultivation of HEK293 Cells and CHO Cell Lines in Suspension

5. Transfection Methods

6. Conclusions

Acknowledgments

References

Chapter 16: Tagging for Protein Expression

1. Introduction

2. Some Considerations When Designing a Tagged Protein

3. Protein Affinity Tags

4. Solubility Tags

5. Removal of Tags

6. Conclusions

Acknowledgment

References

Chapter 17: Refolding Solubilized Inclusion Body Proteins

1. Introduction

2. General Refolding Consideration

3. General Procedures

4. General Protocol

5. Comments on this General Procedure

6. Performing a Protein Refolding Test Screen

7. Other Refolding Procedures

8. Refolding Database: Refold

9. Strategies to Increase Proportion of Soluble Protein

10. Conclusion

References

Section 4: Preparation of extracts and subcellular fractionation

Chapter 18: Advances in Preparation of Biological Extracts for Protein Purification

1. Introduction

2. Chemical and Enzymatic Cell Disruption

3. Mechanical Cell Disruption

4. Concluding Remarks

5. Procedures, Reagents, and Tips for Cell Disruption

References

Chapter 19: Isolation of Subcellular Organelles and Structures

1. Introduction

2. Extraction and Prefractionation of Subproteomes

References

Section 5: Purification Procedures: Bulk Methods

Chapter 20: Protein Precipitation Techniques

1. Introduction

2. Ammonium Sulfate Precipitation

3. Polyethyleneimine Precipitation

4. Other Methods

5. General Procedures When Fractionating Proteins by Precipitation

References

Chapter 21: Affi-Gel Blue for Nucleic Acid Removal and Early Enrichment of Nucleotide Binding Proteins

1. A Representative Protocol

References

Section 6: Purification Procedures: Chromatographic Methods

Chapter 22: Ion-Exchange Chromatography

1. Introduction

2. Principle

3. Stationary Phases

4. Binding Conditions

5. Elution Conditions

6. Operation of Ion-Exchange Columns

7. Example: Separation of Complex Protein Mixture

8. Example: High-Resolution Separation with a Monolithic Column

References

Chapter 23: Gel Filtration

1. Principle

2. Practice

Chapter 24: Protein Chromatography on Hydroxyapatite Columns

1. Introduction

2. Mechanisms

3. Chemical Characteristics

4. Purification Protocol Development

5. Packing Laboratory-Scale Columns

6. Process-Scale Column Packing

7. Applications

References

Chapter 25: Theory and Use of Hydrophobic Interaction Chromatography in Protein Purification Applications

1. Theory

2. Latest Technology in HIC Adsorbents

3. Procedures for Use of HIC Adsorbents

References

Section 7: Purification Procedures: Affinity Methods

Chapter 26: Affinity Chromatography: General Methods

1. Introduction

2. Selection of Affinity Matrix

3. Selection of Ligands

4. Attachment Chemistry

5. Purification Method

References

Chapter 27: Immobilized-Metal Affinity Chromatography (IMAC): A Review

1. Overview on IMAC Ligands and Immobilized Ions

2. IMAC Applications

3. Conclusions

Acknowledgments

References

Chapter 28: Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography

1. Introduction

2. Polyol-Responsive Monoclonal Antibodies

3. Conclusions

Disclosure

References

Section 8: Purification Procedures: Electrophoretic Methods

Chapter 29: One-Dimensional Gel Electrophoresis

1. Background

2. Polyacrylamide Gels

3. Principle of Method

4. Procedure

5. Detection of Proteins in Gels

6. Marker Proteins

7. Molecular Weight Determination

8. Preparative Electrophoresis

References

Chapter 30: Isoelectric Focusing and Two-Dimensional Gel Electrophoresis

1. Introduction

2. Materials

3. Methods

References

Chapter 31: Protein Gel Staining Methods: An Introduction and Overview

1. Introduction

2. General Considerations

3. Instrumentation: Detection and Documentation

4. Total Protein Detection

5. Phosphoprotein Detection

6. Glycoprotein Detection

References

Chapter 32: Elution of Proteins from Gels

1. Introduction

2. Elution of Proteins from Gels by Diffusion

3. Replacing the SDS Gel with a Reverse Phase HPLC

4. Electrophoretic Elution

5. Conclusion

References

Chapter 33: Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection

1. Western Blotting

2. Types of Western Blots

3. Detection Methods

4. The Chemiluminescence Signal

5. Common Problems and their Explanations

6. Blotting and Optimization Protocols using Chemiluminescent Substrates

References

Section 9: Purification Procedures: Membrane Proteins And Glycoproteins

Chapter 34: Detergents: An Overview

1. Introduction

2. Detergent Structure

3. Properties of Detergents in Solution

4. Exploiting the Physicochemical Parameters of Detergents for Membrane Protein Purification

5. Detergent Removal and Detergent Exchange

6. Choosing the Right Detergent

7. Conclusions

Acknowledgments

References

Chapter 35: Purification of Membrane Proteins

1. Introduction

2. Preparation of Membranes

3. Solubilization of Native Membrane Proteins

4. Purification of Membrane Proteins

5. Detergent Removal and Detergent Exchange

6. Expression and Purification of Recombinant Integral Membrane Proteins

References

Chapter 36: Purification of Recombinant G-Protein-Coupled Receptors

1. Introduction

2. Solubilization: General Considerations

3. Purification: General Considerations

4. Solubilization and Purification of a Recombinant Neurotensin Receptor NTS1

5. Analysis of Purified NTS1

6. Conclusions

Acknowledgments

References

Chapter 37: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes

1. Introduction

2. Overview of Cell-Free Translation

3. Expression Vectors

4. Gene Cloning

5. PCR Product Cleanup

6. Flexi Vector and PCR Product Digestion Reaction

7. Ligation Reaction

8. Transformation Reaction

9. Purification of Plasmid DNA

10. Preparation of mRNA

11. Preparation of Liposomes

12. Wheat Germ Translation Reaction

13. Purification by Density Gradient Ultracentrifugation

14. Characterization of Proteoliposomes

15. Considerations for Scale-Up

16. Isotopic Labeling for Structural Studies

17. Conclusions

Acknowledgments

References

Section 10: Characterization of Purified Proteins

Chapter 38: Determination of Protein Purity

1. Composition-Based and Activity-Based Analyses

2. Electrophoretic Methods

3. Chromatographic Methods

4. Sedimentation Velocity Methods

5. Mass Spectrometry Methods

6. Light Scattering Methods

References

Chapter 39: Determination of Size, Molecular Weight, and Presence of Subunits

1. Introduction

2. Chemical Methods

3. Transport Methods

4. Scattering Methods

5. Presence of Subunits

References

Chapter 40: Identification and Quantification of Protein Posttranslational Modifications

1. Introduction

2. Enrichment Techniques for Identifying PTMs

3. Nitrosative Protein Modifications

4. Methylation and Acetylation

5. Mass Spectrometry Analysis

6. CID versus ECD versus ETD

7. Quantifying PTMs

8. Future Directions

Acknowledgments

References

Section 11: Additional Techniques

Chapter 41: Parallel Methods for Expression and Purification

1. Introduction

2. Strategies Based on End-Use

3. Parallel Cloning Strategies for Creating Expression Constructs

4. Small-Scale Expression Screening to Identify Suitable Constructs

5. Analytical Testing of Proteins for Selection

6. Large-Scale Parallel Expression

7. Conclusion

Acknowledgments

References

Chapter 42: Techniques to Isolate O2-Sensitive Proteins: [4Fe–4S]-FNR as an Example

1. Introduction

2. Anaerobic Isolation of 4Fe-FNR

3. Characterization of [4Fe-4S]2+ Cluster Containing FNR

4. Summary

References

Section 12: Concluding Remarks

Chapter 43: Rethinking Your Purification Procedure

1. Introduction

Chapter 44: Important but Little Known (or Forgotten) Artifacts in Protein Biochemistry

1. Introduction

2. SDS Gel Electrophoresis Sample Preparation

3. Buffers

4. Chromatography

5. Protein Absorption During Filtration

6. Chemical Leaching from Plasticware

7. Cyanate in Urea

References

Author Index

Subject Index

Color Plates