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Production of Membrane Proteins Strategies for Expression and Isolation 1st Edition by Anne Skaja Robinson ISBN 3527327290 9783527327294

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Production of Membrane Proteins Strategies for Expression and Isolation 1st Edition Anne Skaja Robinson Digital Instant Download

Author(s): Anne Skaja Robinson
ISBN(s): 9783527327294, 3527327290
Edition: 1
File Details: PDF, 3.89 MB
Year: 2011
Language: english
SKU: EB-2514096 Category: Tag:

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ISBN 10: 3527327290
ISBN 13: 9783527327294
Author: Anne Skaja Robinson

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.

Production of Membrane Proteins Strategies for Expression and Isolation 1st Table of contents:

PART I: Expression Systems

1. 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

2. MEMBRANE PROTEIN EXPRESSION IN SACCHAROMYCES CEREVISIAE

  • Introduction

  • Getting Started

  • Special Considerations

  • Case Studies

  • Conclusions

3. 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

4. HETEROLOGOUS PRODUCTION OF ACTIVE MAMMALIAN G-PROTEIN-COUPLED RECEPTORS USING BACULOVIRUS-INFECTED INSECT CELLS

  • Introduction

  • Experimental

  • Conclusions and Future Perspectives

5. MEMBRANE PROTEIN EXPRESSION IN MAMMALIAN CELLS

  • Introduction

  • Mammalian Systems

  • Case Studies

  • Conclusions

6. 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

7. PERIPHERAL MEMBRANE PROTEIN PRODUCTION FOR STRUCTURAL AND FUNCTIONAL STUDIES

  • Introduction

  • Case Studies of Peripheral Membrane Proteins

  • Conclusions

8. 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

9. STRUCTURAL BIOLOGY OF MEMBRANE PROTEINS

  • Introduction

  • Folding and Structural Analysis of Membrane Proteins

  • Test Cases

PART III: Emerging Methods and Approaches

10. ENGINEERING INTEGRAL MEMBRANE PROTEINS FOR EXPRESSION AND STABILITY

  • Introduction

  • Engineering Higher Expression

  • Engineering Higher Stability

  • Conclusions

11. 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

12. SOLUBILIZATION, PURIFICATION, AND CHARACTERIZATION OF INTEGRAL MEMBRANE PROTEINS

  • Introduction

  • Solubilization of IMPs

  • IMP Purification

  • Characterization of Solubilized IMPs

13. 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

14. RAPID OPTIMIZATION OF MEMBRANE PROTEIN PRODUCTION USING GREEN FLUORESCENT PROTEIN-FUSIONS AND LEMO21(DE3)

  • Introduction

  • Main Protocol

  • Materials

  • Expression and Isolation of GFP-His8

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Tags: Anne Skaja Robinson, Membrane Proteins, Isolation