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基本説明
Contains step-by-step protocols for every methods.
Full Description
Designed as a research-level guide to current strategies and methods of membrane protein production on the small to intermediate scale, this practice-oriented book provides detailed, step-by-step laboratory protocols as well as an explanation of the principles behind each method, together with a discussion of its relative advantages and disadvantages.
Following an introductory section on current challenges in membrane protein production, the book goes on to look at expression systems, emerging methods and approaches, and protein specific considerations.
Case studies illustrate how to select or sample the optimal production system for any desired membrane protein, saving both time and money on the laboratory as well as the technical production scale.
Unique in its coverage of "difficult" proteins with large membrane-embedded domains, proteins from extremophiles, peripheral membrane proteins, and protein fragments.
Contents
Preface
INTRODUCTION
Expression
Solubilization and Structural Methods
Abbreviations
PART I: Expression Systems
BACTERIAL SYSTEMS
Introduction
Understanding the Problem
Vector/Promoter Types
T7 Expression System
Tunable T7 Expression Systems
Other Useful Membrane Protein Expression Strains
Clone Stability
Media Types
Fusion Partners/Membrane Targeting Peptides
Chaperone Overexpression
Cautionary Notes Related to Chaperone Overexpression
Emerging Role of Quality Control Proteases
Tag Selection
Potential Expression Yield
Strategies to Overcome Protein Instability
MEMBRANE PROTEIN EXPRESSION IN SACCHAROMYCES CEREVISIAE
Introduction
Getting Started
Special Considerations
Case Studies
Conclusions
EXPRESSION SYSTEMS: PICHIA PASTORIS
Introduction
A (Brief) Summary on the (Long) History of P. pastoris
Introducing P. pastoris as a Biotechnological Tool: Its (Extended) Strengths and (Limited) Weaknesses
Basics of the P. pastoris Expression System
Successful Large-Scale Expression of Membrane Proteins Using P. pastoris
Guidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as Models
Conclusions and Future Directions
HETEROLOGOUS PRODUCTION OF ACTIVE MAMMALIAN G-PROTEIN-COUPLED RECEPTORS USING BACULOVIRUS-INFECTED INSECT CELLS
Introduction
Experimental
Conclusions and Future Perspectives
MEMBRANE PROTEIN EXPRESSION IN MAMMALIAN CELLS
Introduction
Mammalian Systems
Case Studies
Conclusions
MEMBRANE PROTEIN PRODUCTING USING PHOTOSYNTHETIC BACTERIA: A PRACTICAL GUIDE
Introduction
Preparation of Expression Constructs
Transfer of Plasmid DNA to Rhodobacter via Conjugal Mating
Small-Scale Screening for Expression and Localization of Target Protein in Rhodobacter
Large-Scale Culture
Detergent Solubilization and Chromatographic Purification of Expressed Membrane Proteins
Protein Identification and Assessment of Purity
Preparations of Specialized Rhodobacter Membranes
Appendix: Media and Buffer Formulations
PART II: Protein-Specific Considerations
PERIPHERAL MEMBRANE PROTEIN PRODUCTION FOR STRUCTURAL AND FUNCTIONAL STUDIES
Introduction
Case Studies of Peripheral Membrane Proteins
Conclusions
EXPRESSION OF G-PROTEIN-COUPLED RECEPTORS
Introduction
Bacterial Expression of GPCRs
Expression of GPCrs in Inclusion Bodies and Refolding
Expression of GPCrs in Yeast
Expression of GPCrs in Insect Cells
Expression of GPCrs in Mammalian Cell Lines
Expression of GPCrs in Retina Rod Cells
Expression of GPCrs in a Cell-Free System
Stabilization of GPCrs during Solubilization and Purification
Conclusions
STRUCTURAL BIOLOGY OF MEMBRANE PROTEINS
Introduction
Folding and Structural Analysis of Membrane Proteins
Test Cases
PART III: Emerging Methods and Approaches
ENGINEERING INTEGRAL MEMBRANE PROTEINS FOR EXPRESSION AND STABILITY
Introduction
Engineering Higher Expression
Engineering Higher Stability
Conclusions
EXPRESSION AND PURIFICATION OF G-PROTEIN-COUPLED RECEPTORS FOR NUCLEAR MAGNETIC RESONANCE STRUCTURAL STUDIES
Introduction: G-Protein-Coupled Receptor Superfamily
CXCR1
GPCR Structures
NMR Studies of GPCRs
Expression Systems
Cloning of CXCR1 into pGEX2a
Expression of CXCR1
Purification
Refolding and Reconstitution
Binding and Activity Measurements
NMR Spectra
SOLUBILIZATION, PURIFICATION, AND CHARACTERIZATION OF INTEGRAL MEMBRANE PROTEINS
Introduction
Solubilization of IMPs
IMP Purification
Characterization of Solubilized IMPs
STABILIZING MEMBRANE PROTEINS IN DETERGENT AND LIPID SYSTEMS
Introduction
Choice of Detergent: Solubilization versus Stability
Mitigating Protein Denaturation
Making or Selecting a Stable Protein
Conclusions
RAPID OPTIMIZATION OF MEMBRANE PROTEIN PRODUCTION USING GREEN FLUORESCENT PROTEIN-FUSIONS AND LEMO21(DE3)
Introduction
Main Protocol
Materials
Expression and Isolation of GFP-His8
Conclusions
