Production of Membrane Proteins - Strategies for Expression and Isolation

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.

List of contents

PrefaceINTRODUCTIONExpressionSolubilization and Structural MethodsAbbreviationsPART I: Expression SystemsBACTERIAL SYSTEMSIntroductionUnderstanding the ProblemVector/Promoter TypesT7 Expression SystemTunable T7 Expression SystemsOther Useful Membrane Protein Expression StrainsClone StabilityMedia TypesFusion Partners/Membrane Targeting PeptidesChaperone OverexpressionCautionary Notes Related to Chaperone OverexpressionEmerging Role of Quality Control ProteasesTag SelectionPotential Expression YieldStrategies to Overcome Protein InstabilityMEMBRANE PROTEIN EXPRESSION IN SACCHAROMYCES CEREVISIAEIntroductionGetting StartedSpecial ConsiderationsCase StudiesConclusionsEXPRESSION SYSTEMS: PICHIA PASTORISIntroductionA (Brief) Summary on the (Long) History of P. pastorisIntroducing P. pastoris as a Biotechnological Tool: Its (Extended) Strengths and (Limited) WeaknessesBasics of the P. pastoris Expression SystemSuccessful Large-Scale Expression of Membrane Proteins Using P. pastorisGuidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as ModelsConclusions and Future DirectionsHETEROLOGOUS PRODUCTION OF ACTIVE MAMMALIAN G-PROTEIN-COUPLED RECEPTORS USING BACULOVIRUS-INFECTED INSECT CELLSIntroductionExperimentalConclusions and Future PerspectivesMEMBRANE PROTEIN EXPRESSION IN MAMMALIAN CELLSIntroductionMammalian SystemsCase StudiesConclusionsMEMBRANE PROTEIN PRODUCTING USING PHOTOSYNTHETIC BACTERIA: A PRACTICAL GUIDEIntroductionPreparation of Expression ConstructsTransfer of Plasmid DNA to Rhodobacter via Conjugal MatingSmall-Scale Screening for Expression and Localization of Target Protein in RhodobacterLarge-Scale CultureDetergent Solubilization and Chromatographic Purification of Expressed Membrane ProteinsProtein Identification and Assessment of PurityPreparations of Specialized Rhodobacter MembranesAppendix: Media and Buffer FormulationsPART II: Protein-Specific ConsiderationsPERIPHERAL MEMBRANE PROTEIN PRODUCTION FOR STRUCTURAL AND FUNCTIONAL STUDIESIntroductionCase Studies of Peripheral Membrane ProteinsConclusionsEXPRESSION OF G-PROTEIN-COUPLED RECEPTORSIntroductionBacterial Expression of GPCRsExpression of GPCrs in Inclusion Bodies and RefoldingExpression of GPCrs in YeastExpression of GPCrs in Insect CellsExpression of GPCrs in Mammalian Cell LinesExpression of GPCrs in Retina Rod CellsExpression of GPCrs in a Cell-Free SystemStabilization of GPCrs during Solubilization and PurificationConclusionsSTRUCTURAL BIOLOGY OF MEMBRANE PROTEINSIntroductionFolding and Structural Analysis of Membrane ProteinsTest CasesPART III: Emerging Methods and ApproachesENGINEERING INTEGRAL MEMBRANE PROTEINS FOR EXPRESSION AND STABILITYIntroductionEngineering Higher ExpressionEngineering Higher StabilityConclusionsEXPRESSION AND PURIFICATION OF G-PROTEIN-COUPLED RECEPTORS FOR NUCLEAR MAGNETIC RESONANCE STRUCTURAL STUDIESIntroduction: G-Protein-Coupled Receptor SuperfamilyCXCR1GPCR StructuresNMR Studies of GPCRsExpression SystemsCloning of CXCR1 into pGEX2aExpression of CXCR1PurificationRefolding and ReconstitutionBinding and Activity MeasurementsNMR SpectraSOLUBILIZATION, PURIFICATION, AND CHARACTERIZATION OF INTEGRAL MEMBRANE PROTEINSIntroductionSolubilization of IMPsIMP PurificationCharacterization of Solubilized IMPsSTABILIZING MEMBRANE PROTEINS IN DETERGENT AND LIPID SYSTEMSIntroductionChoice of Detergent: Solubilization versus StabilityMitigating Protein DenaturationMaking or Selecting a Stable ProteinConclusionsRAPID OPTIMIZATION OF MEMBRANE PROTEIN PRODUCTION USING GREEN FLUORESCENT PROTEIN-FUSIONS AND LEMO21(DE3)IntroductionMain ProtocolMaterialsExpression and Isolation of GFP-His8Conclusions

About the author

Anne Skaja Robinson is Professor and Associate Chair for Biochemical Engineering in the Department of Chemical Engineering at the University of Delaware in Newark, Delaware, USA. She obtained her Bachelor's and Master's degrees from The Johns Hopkins University, and her Ph.D. from the University of Illinois at Urbana-Champaign. Prior to joining the faculty at Delaware in 1997, she was a postdoctoral fellow in the Department of Biology at MIT. She is a widely acknowledged leader in the field of protein production, and has authored over 50 scientific publications. Also, she is the recipient of numerous awards, including NSF's PECASE/Career award, the DuPont Young Professor award, and is an 2011 AIMBE Fellow. She is a member of the Editorial Boards of Biotechnology and Bioengineering, and Biotechnology Journal, and the former Chair of the American Chemical Society BIOT division.

Summary

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.

Product details

• Authors: Anne Skaja Robinson
• Assisted by: Anne S. Robinson (Editor), Anne Skaja Robinson (Editor), Ann Skaja Robinson (Editor), Anne Skaja Robinson (Editor)
• Publisher: Wiley-VCH
• Languages: English, German
• Product format: Hardback
• Released: 21.06.2011
• EAN: 9783527327294
• ISBN: 978-3-527-32729-4
• No. of pages: 420
• Dimensions: 177 mm x 248 mm x 26 mm
• Weight: 988 g
• Illustrations: 50 SW-Abb., 9 Farbabb., 21 Tabellen
• Subjects: Natural sciences, medicine, IT, technology > Chemistry > Organic chemistry; Chemie, Molekularbiologie, Proteine, Life Sciences, Biowissenschaften, Membran, molecular biology, biotechnology, chemistry, Protein Science, Wirkstoffforschung u. -entwicklung, Drug Discovery & Development, Biotechnologie i. d. Chemie