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Production of Membrane Proteins: Strategies for Expression and Isolation
Anne Skaja Robinson (Editor)
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Últimas novedades
química general
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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.
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Preface.
List of Contributors.
Introduction (Anne Skaja Robinson and Patrick J. Loll).
Part One Expression Systems.
1 Bacterial Systems (James Samuelson).
1.1 Introduction.
1.2 Understanding the Problem.
1.3 Vector/Promoter Types.
1.4 T7 Expression System.
1.5 Tunable T7 Expression Systems.
1.6 Other Useful Membrane Protein Expression Strains.
1.7 Clone Stability.
1.8 Media Types.
1.9 Fusion Partners/Membrane Targeting Peptides.
1.10 Chaperone Overexpression.
1.11 Cautionary Notes Related to Chaperone Overexpression.
1.12 Emerging Role of Quality Control Proteases.
1.13 Tag Selection.
1.14 Potential Expression Yield.
1.15 Strategies to Overcome Protein Instability.
2 Membrane Protein Expression in Saccharomyces cerevisiae (Zachary Britton, Carissa Young, Özge Can, Patrick McNeely, Andrea Naranjo, and Anne Skaja Robinson).
2.1 Introduction.
2.2 Getting Started.
2.3 Special Considerations.
2.4 Case Studies.
2.5 Conclusions.
3 Expression Systems: Pichia pastoris (Fatima Alkhalfi oui, Christel Logez, Olivier Bornert, and Renaud Wagner).
3.1 Introduction.
3.2 A (Brief) Summary on the (Long) History of P. pastoris.
3.3 Introducing P. pastoris as a Biotechnological Tool: Its (Extended) Strengths and (Limited) Weaknesses.
3.4 Basics of the P. pastoris Expression System.
3.5 Successful Large-Scale Expression of Membrane Proteins Using P. pastoris.
3.6 Guidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as Models.
3.7 Conclusions and Future Directions.
4 Heterologous Production of Active Mammalian G-Protein-Coupled Receptors Using Baculovirus-Infected Insect Cells (Mark Chiu, Brian Estvander, Timothy Esbenshade, Steve Kakavas, Kathy Krueger, Marc Lake, and Ana Pereda-Lopez).
4.1 Introduction.
4.2 Experimental.
4.3 Conclusions and Future Perspectives.
5 Membrane Protein Expression in Mammalian Cells (Deniz B. Hizal, Erika Ohsfeldt, Sunny Mai, and Michael J. Betenbaugh).
5.1 Introduction.
5.2 Mammalian Systems.
5.3 Case Studies.
5.4 Conclusions.
6 Membrane Protein Production Using Photosynthetic Bacteria: A Practical Guide (Philip D. Laible, Donna L. Mielke, and Deborah K. Hanson).
6.1 Introduction.
6.2 Preparation of Expression Constructs.
6.3 Transfer of Plasmid DNA to Rhodobacter via Conjugal Mating.
6.4 Small-Scale Screening for Expression and Localization of Target Protein in Rhodobacter.
6.5 Large-Scale Culture.
6.6 Detergent Solubilization and Chromatographic Purification of Expressed Membrane Proteins.
6.7 Protein Identifi cation and Assessment of Purity.
6.8 Preparations of Specialized Rhodobacter Membranes.
Part Two Protein-Specifi c Considerations.
7 Peripheral Membrane Protein Production for Structural and Functional Studies (Brian J. Bahnson).
7.1 Introduction.
7.2 Case Studies of Peripheral Membrane Proteins.
7.3 Conclusions.
8 Expression of G-Protein-Coupled Receptors (Alexei Yeliseev and Krishna Vukoti).
8.1 Introduction.
8.2 Bacterial Expression of GPCRs.
8.3 Expression of GPCRs in Inclusion Bodies, and Refolding.
8.4 Expression of GPCRs in Yeast.
8.5 Expression of GPCRs in Insect Cells.
8.6 Expression of GPCRs in Mammalian Cell Lines.
8.7 Expression of GPCRs in Retina Rod Cells.
8.8 Expression of GPCRs in a Cell-Free System.
8.9 Stabilization of GPCRs during Solubilization and Purification.
8.10 Conclusions.
9 Structural Biology of Membrane Proteins (David Salom and Krzysztof Palczewski).
9.1 Introduction.
9.2 Folding and Structural Analysis of Membrane Proteins.
9.3 Test Cases.
Part Three Emerging Methods and Approaches.
10 Engineering Integral Membrane Proteins for Expression and Stability (Igor Dodevski and Andreas Plückthun).
10.1 Introduction.
10.2 Engineering Higher Expression.
10.3 Engineering Higher Stability.
10.4 Conclusions.
11 Expression and Purifi cation of G-Protein-Coupled Receptors for Nuclear Magnetic Resonance Structural Studies (Fabio Casagrande, Klaus Maier, Hans Kiefer, Stanley J. Opella, and Sang Ho Park).
11.1 Introduction: G-Protein-Coupled Receptor Superfamily.
11.2 CXCR1.
11.3 GPCR Structures.
11.4 NMR Studies of GPCRs.
11.5 Expression Systems.
11.6 Cloning of CXCR1 into pGEX2a.
11.7 Expression of CXCR1.
11.8 Purification.
11.9 Refolding and Reconstitution.
11.10 Binding and Activity Measurements.
12 Solubilization, Purifi cation, and Characterization of Integral Membrane Proteins (Víctor Lórenz-Fonfría, Alex Perálvarez-Marín, Esteve Padrós, and Tzvetana Lazarova).
12.1 Introduction.
12.2 Solubilization of IMPs.
12.3 IMP Purification.
12.4 Characterization of Solubilized IMPs.
13 Stabilizing Membrane Proteins in Detergent and Lipid Systems (Mark Lorch and Rebecca Batchelor).
13.1 Introduction.
13.2 Choice of Detergent: Solubilization versus Stability.
13.3 Mitigating Protein Denaturation.
13.4 Making or Selecting a Stable Protein.
13.5 Conclusions.
14 Rapid Optimization of Membrane Protein Production Using Green Fluorescent Protein-Fusions and Lemo21(DE3) (Susan Schlegel, Mirjam Klepsch, Dimitra Gialama, David Wickström, David Drew, and Jan-Willem de Gier).
14.1 Introduction.
14.2 Main Protocol.
14.3 Materials.
14.4 Expression and Isolation of GFP-His8.
14.5 Conclusions.
Index.
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