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THÔNG TIN TÀI LIỆU
Cấu trúc
Preface
Contents
Contributors
Part I: High-Throughput Cloning, Expression Screening, and Optimization
Chapter 1: Recombinant Protein Expression in E. coli: A Historical Perspective
1 Introduction
2 Expression from E. coli RNAP Promoters
3 Maximizing Expression Levels
4 Fusion Tags
5 The Protein Is the Most Important Variable
6 High-Throughput Methods
7 Heteromeric Complexes
8 One Method Fits All?
9 Future Prospects
References
Chapter 2: N- and C-Terminal Truncations to Enhance Protein Solubility and Crystallization: Predicting Protein Domain Boundaries with Bioinformatics Tools
1 Introduction
2 Materials
3 Methods
3.1 Prediction of Protein Secondary Structure and Domains Using Sequence and Structural Homology
3.1.1 Domain Prediction Using Homology Searching: BLAST and the CDD
3.1.2 Domain Prediction with HMM Databases: SMART and PFAM
3.1.3 The PSIPRED Workbench for Protein Domain and Secondary Structure Prediction
3.2 Protein Domain Identification Using Globularity and Disorder Prediction
3.2.1 Disorder Analysis with FoldIndex
3.2.2 Disorder Analysis with GlobPlot
3.3 Combining Bioinformatics Approaches for Domain Boundary Prediction
3.4 Further Methods for Domain Boundary Analysis: Beyond Bioinformatics
4 Notes
References
Chapter 3: Harnessing the Profinity eXact™ System for Expression and Purification of Heterologous Proteins in E. coli
1 Introduction
2 Materials
2.1 Bacterial Strains and DNA Cloning
2.2 Profinity eXact™ Expression Vectors
2.3 Protein Expression
2.4 Cell Extraction, Protein Purification and Analysis
2.5 Column Regeneration
3 Methods
3.1 Establishment of Expression Vectors
3.2 Protein Expression
3.3 Protein Extraction, Purification, and Analysis
3.3.1 Preparation of Chromatography Resin
3.3.2 Cell Extraction by Sonication
3.3.3 Protein Purification and Analysis
3.3.4 Resin Regeneration
4 Notes
References
Chapter 4: ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences
1 Introduction
2 Materials
2.1 Library Construction
2.2 Small-Scale Expression Screening
3 Methods
3.1 Cloning of Target Insert into pESPRIT002
3.2 Preparation of High-Quality, Nick-Free Plasmid DNA
3.3 Vector Linearization, Truncation, and End-Polishing
3.7 Identification of Potentially Soluble Clones from the Random Truncation Library
3.8 Purification Screening and Characterization of Soluble Clones
4 Notes
References
Chapter 5: Optimizing Expression and Solubility of Proteins in E. coli Using Modified Media and Induction Parameters
1 Introduction
2 Materials
2.1 Construction of Expression Clones
2.2 Generation of E. coli Expression Strains
2.3 E. coli Protein Expression
2.4 Cell Lysis and Analysis
3 Methods
3.1 Construction of Expression Clones
3.2 Generation of E. coli Expression Strains
3.3 E. coli Protein Expression
3.4 Cell Lysis and Analysis
3.5 Optimization of Expression Conditions
4 Notes
References
Chapter 6: Optimization of Membrane Protein Production Using Titratable Strains of E. coli
1 Introduction
1.1 T7 RNA Polymerase-Driven Expression of Heterologous Proteins
1.2 Small-Molecule-Driven Fine-Tuning of Expression Levels
1.3 Bi-Variate Analysis of Protein Expression
1.4 KRX: Titration of T7 RNA Polymerase Transcription
1.5 Inhibition of T7 RNA Polymerase Activity Using Titratable T7 Lysozyme
1.6 BL21(IL3) Post-transcriptional Control of Heterologous Protein Expression
2 Materials
2.1 Vectors
2.2 Small-Scale E. coli Expression Screen
2.2.1 Transformation, Growth and Induction of Cultures
2.2.2 Harvesting and Processing of Cultures
2.3 Measurement of Relative Fluorescence Units (RFU) and Optical Density (OD)
2.4 In-Gel Fluorescence
3 Methods
3.1 Transformation into E. coli Expression Strains
3.2 Preparation of Overnight Cultures
3.3 Growth and Induction of Cultures Using 24-Well Deep Well Blocks (See Note 6)
3.3.1 Lemo21(DE3)
3.3.2 KRX (See Note 9)
3.3.3 IL3
3.4 Growth and Induction of Cultures Using 96-Well Deep Well Blocks (See Note 10)
3.5 Harvesting of Cultures
3.5.1 24-Well Blocks
3.5.2 96-Well Blocks
3.6 Measurement of Relative Fluorescence Units (RFU) and Optical Density (OD) and Calculation of RFU/OD
3.7 GFP In-Gel Fluorescence
4 Notes
References
Chapter 7: Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) or pReX and GFP-Fusions
1 Introduction
1.1 The Escherichia coli Lemo21(DE3) Strain and the pReX Expression Vector
1.2 Membrane Protein GFP-Fusions
2 Materials
2.1 Culturing of Cells
2.2 Monitoring Production of Membrane Protein GFP-Fusions
2.3 Membrane and GFP Isolation
3 Methods
3.1 Determination of Membrane Protein Topology
3.2 Selection of Expression Vector
3.3 Identification of the Optimal Production Conditions
3.3.1 Transformation and Culturing
3.3.2 Whole Cell Fluorescence
3.3.3 In-Gel Fluorescence
3.3.4 Monitoring the Ratio of Membrane Inserted to Non-inserted Membrane Protein
3.4 GFP-His8 as a Reference for Whole Cell Fluorescence and In-Gel Fluorescence Measurements
3.5 Scaling Up Membrane Protein Production
3.5.1 Scaling Up of Cultures
3.5.2 Isolation of Membranes
3.6 mNeonGreen as a Fluorescent Fusion Tag for Monitoring Membrane Protein Production
4 Notes
References
Chapter 8: High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B
1 Introduction
2 Materials
3 Methods
3.1 Preculture
3.2 Protein Expression Culture
4 Notes
References
Part II: Protein Purification and Solubility Enhancement
Chapter 9: A Generic Protocol for Purifying Disulfide-Bonded Domains and Random Protein Fragments Using Fusion Proteins with SUMO3 and Cleavage by SenP2 Protease
1 Introduction
2 Materials
2.1 Expression of HisSenP2 Protease and SUMO3-Fusion Proteins
2.2 Purification of His-Tagged Proteins
3 Methods
3.1 Expression and Purification of HisSenP2 Protease (4 L Total Culture Vol.)
3.2 Expression and Purification of SUMO-Fusion Protein
4 Notes
References
Chapter 10: A Strategy for Production of Correctly Folded Disulfide-Rich Peptides in the Periplasm of E. coli
1 Introduction
2 Materials
2.1 Plasmid Construction
2.2 Transformation
2.3 5 mL Starter Culture
2.4 Glycerol Stocks
2.5 50 mL Starter Culture
2.6 1 L Expression Culture
2.7 Cell Lysis
2.8 Nickel Affinity Purification
2.9 TEV Cleavage
2.10 SDS-PAGE Analysis
2.11 Peptide Purification by Liquid Chromatography
2.12 Quality Control and Quantitation
3 Methods
3.1 Plasmid Construction
3.2 Transformation
3.3 5 mL Starter Culture
3.4 Glycerol Stocks
3.5 50 mL Starter Culture
3.6 1 L Expression Culture
3.7 Cell Lysis
3.8 Nickel Affinity Purification
3.9 TEV Protease Cleavage
3.10 SDS-PAGE Analysis of DRP Expression, Purification, and Cleavage
3.11 Peptide Purification by Liquid Chromatography
3.12 Quality Control and Quantitation
4 Notes
References
Chapter 11: Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter
1 Introduction
2 Materials
2.1 Cloning SteT into the pTET-GFP11 Vector and Generation of SteT Random Mutants
2.2 “In Colony” Split GFP Complementation Assay
2.3 Split GFP Complementation Assay in Liquid Cultures
2.4 Stability of SteT Random Mutants After Detergent Solubilization
2.4.1 Isolation of E. coli Plasma Membranes
2.4.2 FSEC Analysis
3 Methods
3.1 Generating the Library of SteT Random Mutants
3.1.1 Cloning SteT into the pTET-GFP11 Vector and Generation of SteT Random Mutants by Error-Prone PCR
3.1.2 Making E. coli BL21(DE3) Competent Cells Harboring pETGFP1–10
3.1.3 “In Colony” Identification of Mutants Expressing and Inserting in the E. coli Inner Membrane Using the Split GFP Complementation Assay
3.2 Expression Yield of the Mutant Library Quantified Using the Split GFP Assay in Liquid Cultures
3.3 Stability of SteT Random Mutants After Detergent Solubilization
3.3.1 Isolation of E. coli Plasma Membranes
3.3.2 FSEC Analysis of SteT Random Mutants After DDM Solubilization
4 Notes
References
Chapter 12: Acting on Folding Effectors to Improve Recombinant Protein Yields and Functional Quality
1 Introduction
2 Materials
2.1 Vectors
2.2 Small-Scale Protein Production
2.3 Small-Scale Production Output Analysis
2.4 Cytoplasmic Large-Scale Protein Production of Antibody Fragments
2.5 Protein Structural Quality Evaluation
3 Methods
3.1 Vector Preparation
3.2 Small-Scale Protein Production (See Note 7)
3.3 Small-Scale Production Output Analysis: SDS-PAGE (See Note 12)
3.4 Cytoplasmic Large-Scale Recombinant Antibody Production