Production of Membrane Proteins

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