Production of Membrane Proteins (PDF)

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

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.

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