Textbook on Cloning, Expression and Purification of Recombinant Proteins

Preface
Contents
Editor and Contributors
1: A Brief Introduction to Recombinant DNA Technology
1.1 Overview: Recombinant DNA Technology
1.2 Brief History of Recombinant DNA Technology
1.3 Tools in rDNA Technology
1.3.1 Restriction Enzymes in Cloning
1.3.2 Vectors
1.3.3 Competent Host Organism
1.4 Protein Expression and Production in Bacterial Systems
1.5 Important Applications and Future Perspectives of rDNA Technology
1.6 Conclusions
References
2: Cloning and Gene Manipulation
2.1 Introduction
2.2 DNA Libraries
2.2.1 Genomic Library
2.2.1.1 Applications
2.2.2 cDNA Library
2.2.2.1 Construction of a cDNA Library
2.2.2.2 Applications of cDNA Library
2.2.3 Difference Between Genomic and cDNA Library (Table 2.2)
2.3 Polymerase Chain Reaction (PCR)
2.3.1 Background
2.3.2 Components of PCR
2.3.3 PCR Protocol
2.4 Restriction Digestion
2.4.1 Restriction Enzymes (Endonucleases)
2.4.2 Steps and Tips for Restriction Digestion
2.5 Ligation
2.5.1 Introduction
2.5.2 DNA Ligases
2.5.3 Ligation Using Linkers and Adaptors
2.5.4 Standardizing the Ligation Reaction
2.5.5 Steps Involved in Ligation
2.6 Ligation Independent Cloning (LIC)
2.6.1 Background
2.6.2 Protocol for LIC
2.6.3 Advantages
2.7 Choice of Host Cells
2.8 Transformation
2.8.1 Protocol for Transformation
2.9 Colony Screening
2.9.1 Blue-White Colony Screening
2.9.1.1 Background
2.9.1.2 Protocol for Blue-White Colony Screening
2.9.1.3 Limitations
2.9.2 Other Screening Methods
2.9.2.1 Positive Selection System
2.9.2.2 Screening by Plasmid Miniprep and RE (Restriction Enzyme) Digests
2.9.2.3 Colony PCR
2.9.2.4 Sanger Sequencing
2.10 Troubleshooting for Subcloning Experiments (Table 2.5)
2.11 Conclusions
Problems
Multiple Choice Questions
Subjective Questions
References
3: Selection of Cloning and Expression Plasmid Vectors
3.1 Introduction
3.2 Classification
3.3 Cloning: An Overview
3.4 The Need to Choose a Plasmid Vector
3.5 Types of Plasmid Vectors
3.5.1 Plasmids Used for Cloning
3.5.1.1 Criteria for Choosing a Plasmid for Cloning
Size of Insert
Copy Number
Cloning Sites (MCS region)
3.5.1.2 Types of Cloning Plasmids
3.5.2 Expression Plasmid Vectors
3.5.3 Affinity Tags for Protein Purification in Expression Plasmids
3.5.3.1 pGEX Plasmids with GST Tag for Protein Expression
3.5.3.2 pMAL Plasmids with MBP Tag for Protein Expression
3.5.3.3 Duet Vectors
3.5.3.4 Cell-Free Expression Systems
3.5.3.5 Other Expression Systems
3.5.4 Conclusion
Problems
Multiple Choice Questions
Subjective Questions
References
4: Transformation and Protein Expression
4.1 Introduction
4.1.1 Competence and Competent Cell Preparation
4.1.2 Competent Cell Preparation
4.1.3 Chemical Method
4.1.4 Preparing Electrocompetent Cells
4.1.5 Transformation Methods
4.2 Heat-Shock Method
4.2.1 Procedure for Bacterial Transformation Using the Heat-Shock Method
4.2.2 Expected Observations
4.3 Electroporation Method
4.3.1 Procedure for Bacterial Transformation Using the Electroporation Method
4.4 Recombinant Protein Expression in Different Bacterial Systems
4.5 Expression of ``Difficult-to-Fold´´ Proteins in E. coli
4.6 Optimizing Gene Expression
4.7 Protein Production Protocol for Bacteria
4.8 Posttranslational Modifications in Bacterial Expression Systems
4.9 Expression in Yeast Cells
4.10 Conclusions
Problems
Multiple Choice Questions
Subjective Questions
References
5: Introduction to Recombinant Protein Purification
5.1 Introduction
5.2 Databases and Tools to Determine Physicochemical Properties of Protein
5.2.1 Physicochemical Parameters Important in Initial Designing of the Purification Procedure
5.2.1.1 Molecular Weight of the Protein
5.2.1.2 Isoelectric Point, pI
5.2.1.3 Molar Extinction Coefficient/Absorptivity Coefficient
5.2.1.4 Cysteine Content
5.2.1.5 Stability
5.2.1.6 Hydrophobicity
5.2.2 Bioinformatics Resources
5.3 Lysis and Protein Extraction
5.3.1 Source Material for the Protein
5.3.1.1 Extraction Methods
5.3.1.2 Extraction Medium/Lysis Buffer
Buffer Salt and pH
5.3.1.3 Detergents and Chaotropic Agents
5.3.1.4 Reducing Agents
5.3.1.5 Stabilizing Additives
5.3.1.6 Nucleases
5.3.1.7 Protease Inhibitors
5.3.2 Clarification of the Extract
5.3.2.1 Centrifugation
5.3.2.2 Filtration
5.4 Checking Solubility and Designing Purification Strategies
5.4.1 Protein Solubility and Precipitation
5.4.2 Salting-out
5.4.3 Ammonium Sulfate Precipitation
5.4.4 Salting-in
5.4.5 Dealing with Proteins in the Inclusion Bodies
5.5 Overview of Chromatography
5.5.1 Affinity Chromatography
5.5.2 Ion Exchange Chromatography (IEX)
5.5.3 Size Exclusion Chromatography
5.5.4 Concluding Remarks
Problems
Multiple Choice Questions
Subjective Questions
References
6: Protein Purification by Affinity Chromatography
6.1 Introduction
6.2 Types of Tags
6.2.1 Polyhistidine Tag
6.2.2 Glutathione-S-Transferase (GST) Tag
6.2.3 Maltose-Binding Protein (MBP) Tag
6.2.4 Calmodulin-Binding Peptide (CBP) Tag
6.2.5 Streptavidin-Binding Peptide (SBP) Tag
6.3 Types of Affinity Chromatography
6.3.1 Purification of Polyhistidine Tag Protein
6.3.1.1 Binding with the Polyhistidine Tag
6.3.1.2 Components of the Chromatographic Matrix
6.3.1.3 Purification Under Different Conditions
6.3.1.4 Elution
6.3.1.5 Troubleshooting
6.3.2 Purification of GST-Tagged Protein
6.3.2.1 pGEX Vectors and Their Gene Fusion Construct
6.3.2.2 Expression of the Fused Protein
6.3.2.3 Affinity-Based Purification of the GST-Fused Protein
6.3.2.4 Elution and Removal of the GST Tag
6.3.2.5 Troubleshooting
6.3.3 Purification of MBP-Tag Recombinant Proteins
6.3.3.1 Expression of MBP-Tag Protein Using pMAL Vector
6.3.3.2 Binding to the Amylose Affinity Column and Purification
6.3.3.3 Removal of the MBP-Tag Through Proteolytic Cleavage
6.3.3.4 Troubleshooting
6.3.4 Purification of Strep-Tag II Recombinant Proteins
6.3.4.1 Expression of the Strep-Tag II Fused Protein
6.3.4.2 Purification and Elution of the Fused Protein Using StrepTactin Affinity Column
6.3.4.3 Troubleshooting
6.4 Role of Affinity Tags in Identifying Protein-Protein Interactions
6.5 Conclusion
Problems
Multiple choice questions
Subjective questions
References
7: Protein Purification by Ion Exchange Chromatography
7.1 Introduction
7.2 Basic Principles of Ion Exchange Chromatography
7.3 Components and Factors of Ion Exchange Chromatography
7.3.1 Ion Exchange Resins
7.3.2 Capacity
7.3.3 Selection of the Ion Exchange Resins
7.3.3.1 Choice of Anionic and Cationic Exchangers
7.3.3.2 Choice of Strong and Weak Exchangers
7.3.3.3 Choice of Particle Size and Porosity of the Resin Matrix
7.3.4 Buffer
7.3.5 Selection of Buffer
7.3.5.1 Buffer Substance
7.3.5.2 pH of the Buffer
7.3.5.3 Ionic Strength of the Buffer
7.3.5.4 Temperature of the Buffer
7.4 Protein Purification Using Ion Exchange Chromatography
7.4.1 Equilibration
7.4.2 Loading of Sample
7.4.3 Washing
7.4.4 Elution
7.4.5 Regeneration
7.5 Instrumentation for Ion Exchange Chromatography
7.5.1 Pump
7.5.2 Injector
7.5.3 Guard Column
7.5.4 Column
7.5.5 Suppressor
7.5.6 Detectors
7.5.7 Fraction Collector
7.5.8 Data Processing System
7.6 Protocol for Recombinant Protein Purification
7.6.1 Instruments and Materials
7.6.2 Procedure
7.6.2.1 Step 1 (Equilibration of the Column)
7.6.2.2 Step 2 (Binding of the Protein Sample)
7.6.2.3 Step 3 (Removal of Unbound Proteins)
7.6.2.4 Step 4 (Elution of the Bound Protein)
7.6.2.5 Step 5 (Storage of the Protein)
7.6.2.6 Step 6 (Regeneration of the Column)
7.7 Choice of Different Combination of Chromatographic Techniques
7.8 Advantages and Disadvantages of Ion Exchange Chromatography
7.8.1 Advantages
7.8.2 Disadvantages
7.9 Applications of Ion Exchange Chromatography
7.9.1 Purification of Recombinant Proteins
7.9.2 Purification of Enzymes
7.9.3 Miscellaneous Applications
7.10 Troubleshooting
7.11 Conclusion
Problems
Multiple Choice Questions
Subjective Questions
References
8: Gel Filtration Chromatography
8.1 Introduction
8.2 Instrumentation
8.2.1 Pump
8.2.2 Injector
8.2.3 Column
8.2.4 Detector
8.2.5 Fraction Collector
8.2.6 Data Processing System
8.3 Principle of Macromolecular Separation Using Gel Filtration Chromatography
8.4 Choice of Matrix in Gel Filtration Chromatography
8.5 Resolution of Gel Filtration Chromatography
8.5.1 Parameters Affecting Resolution
8.5.1.1 Column Parameters
8.5.1.2 Packing the Column
8.5.1.3 Air Bubbles, Uneven Packing, and Cracks
8.5.1.4 Choice of Eluent
8.5.1.5 Effect of Flow Rate
8.5.1.6 Column Cleaning and Storage
8.5.1.7 Sample Preparation
8.6 Applications of Gel Filtration Chromatography
8.6.1 Molecular Weight Determination
8.6.1.1 Operating Procedure
GFC System
Reagents
Standard Operating Tools
Running the Experiment-Instrumental Setup
Running Analyte Through the GFC Column
8.6.2 Purification of Recombinant Proteins
8.6.3 Desalting and Buffer Exchange
8.6.4 Miscellaneous Applications
8.7 Troubleshooting Tips for Running GFC
8.8 Conclusions
Problems
Multiple Choice Question
Subjective Question
References
9: Protein Purification by Reversed Phase Chromatography and Hydrophobic Interaction Chromatography
9.1 Introduction
9.2 Principle of Surface Adsorption Chromatography
9.3 Reversed Phase Chromatography
9.3.1 Principle of Protein Separation in RPC
9.3.2 Overview of Steps in RPC
9.3.2.1 Column Equilibration
9.3.2.2 Binding of the Extract on Column (Capture)
9.3.2.3 Elution of Protein Molecules (Desorption)
9.3.2.4 Cleaning of Column and Storage
9.3.3 Protocol for Protein Purification Using RPC
9.3.4 Some Important Factors that Govern Optimum Separation and Resolution in RPC
9.3.4.1 Length of the Separation Column
9.3.4.2 Flow Rate
9.3.4.3 Temperature
9.3.4.4 Mobile Phase
9.3.4.5 Gradient Elution
9.3.4.6 Retention of Proteins
9.3.5 Different Uses of RPC
9.3.5.1 Desalting and Protein Concentration
9.3.5.2 High Resolution Separations
9.3.5.3 Large Scale Preparative Purification
9.3.6 Applications
9.4 Hydrophobic Interaction Chromatography
9.4.1 Principle of Hydrophobic Interactions
9.4.2 Difference between RPC and HIC
9.4.3 Principle of HIC
9.4.4 Some Important Factors that Govern the Optimum Separation and Resolution in HIC
9.4.4.1 Stationary Phase
9.4.4.2 Matrix
9.4.4.3 Mobile Phase
9.4.4.4 pH
9.4.4.5 Temperature
9.4.4.6 Additives
9.4.5 Sample Preparation for HIC
9.4.6 Overview of Important Steps in HIC
9.4.6.1 Equilibration of HIC Column
9.4.6.2 Protein Loading
9.4.6.3 Elution of the Bound Protein
9.4.6.4 Column Regeneration
9.4.7 Standard Protocol for HIC
9.4.8 Applications
9.4.8.1 HIC in Combination with Ion Exchange Chromatography
9.4.8.2 HIC in Combination with Gel Filtration Chromatography
9.4.8.3 HIC for Studying Changes in Protein Conformation
9.4.9 Recent Modifications and Improvements in HIC
9.4.9.1 Dual Salt Load Conditioning
9.4.9.2 Improved Resins
9.4.9.3 Membrane HIC
9.4.9.4 Flow Through Mode
9.4.10 Advantages of Using HIC
9.5 Conclusion
9.6 Troubleshooting for RPC and HIC
Problems
Multiple Choice Questions
Subjective Questions
References
10: Purification of Difficult Proteins
10.1 Introduction
10.2 Types of Challenging Proteins and their Purification
10.2.1 Membrane Proteins
10.2.1.1 Overview
Peripheral Proteins
Amphitropic Proteins
Lipid-linked Proteins
Integral Membrane Proteins
10.2.1.2 Problems Encountered
10.2.1.3 Conventional Strategies Employed for Purification
Source
Expression Systems
Prokaryotic Cells
Eukaryotic Cells
Cell-free (CF) Expression System
Summary of the Expression Systems
Techniques for Purification
Sequences, Tags, and Cleavage Sites
Promoters and Plasmids
Antibiotic and Drug-based Screening
Culture Growth Conditions
Detergents and Buffers
10.2.2 Toxic Proteins
10.2.2.1 Overview
10.2.2.2 Problems Encountered
10.2.2.3 Conventional Strategies Employed for Purification
Plasmid Stability/Toxicity Test
Expression Hosts
Construct Design
Promoters, Plasmids, and Tags
Culture Conditions
10.2.3 Inclusion Bodies
10.2.3.1 Overview
10.2.3.2 Problems Encountered
10.2.3.3 Conventional Strategies Employed for Purification
Cell Disruption Isolation and Solubilization
Refolding
10.3 Case Studies of Challenging Proteins
10.3.1 Purification of the Recombinantly Expressed Membrane Protein Ammonium Transporter (AmtB) from E. coli
10.3.1.1 Materials
10.3.1.2 Method
10.3.2 Extraction of Proteins from Inclusion Bodies in E. coli
10.3.2.1 Materials
10.3.2.2 Method
10.4 Conclusion
Problems
Multiple Choice Questions
Subjective Questions
References
11: Protein Quantitation and Detection
11.1 Introduction
11.2 Types of Protein Quantitation Assays
11.2.1 Different Types of Protein Quantitation Assays
11.2.1.1 Dye-Based Assays
11.2.1.2 Bradford (Coomassie Blue) Assay
Principle
Protocol
Experimental Procedure
11.2.1.3 Lowry Assay
Principle
Reagents Required
Solution Preparatory Steps
Experimental Procedure
11.2.1.4 Bicinchoninic Acid (BCA) Assay
Principle
Reagents
Solution Preparatory Steps
Experimental Procedure
11.3 Assays Involving Ultra Violet (UV) Absorption Spectroscopy
11.3.1 UV Absorption Using Micro-Volume Spectroscopy
11.4 Troubleshooting for Protein Quantitation
11.5 Purity Analysis of Proteins of Interest
11.5.1 SDS PAGE
11.5.2 SDS-Page Gel Preparation
Problems
Multiple Choice Questions
Subjective Questions
References
Answer Key
Chapter 2: Cloning and Gene Manipulation
Chapter 3: Selection of Cloning and Expression Plasmid Vectors
Chapter 4: Transformation and Protein Expression
Chapter 5: Introduction to Recombinant Protein Purification
Chapter 6: Protein Purification by Affinity Chromatography
Chapter 7: Protein Purification by Ion Exchange Chromatography
Chapter 8: Gel Filtration Chromatography
Chapter 9: Protein Purification by Reversed Phase Chromatography and Hydrophobic Interaction Chromatography
Chapter 10: Purification of Difficult Proteins
Chapter 11: Protein Quantitation and Detection