The Resource Production of membrane proteins : strategies for expression and isolation, edited by Anne Skaja Robinson

Production of membrane proteins : strategies for expression and isolation, edited by Anne Skaja Robinson

Label
Production of membrane proteins : strategies for expression and isolation
Title
Production of membrane proteins
Title remainder
strategies for expression and isolation
Statement of responsibility
edited by Anne Skaja Robinson
Contributor
Subject
Language
eng
Cataloging source
NLM
Dewey number
572.696
Illustrations
illustrations
Index
index present
LC call number
QP552.M44
LC item number
P76 2011
Literary form
non fiction
Nature of contents
bibliography
NLM call number
  • 2011 C-870
  • QU 55.7
http://library.link/vocab/relatedWorkOrContributorDate
1966-
http://library.link/vocab/relatedWorkOrContributorName
Robinson, Anne Skaja
http://library.link/vocab/subjectName
  • Membrane proteins
  • Membrane Proteins
  • Membrane Proteins
Label
Production of membrane proteins : strategies for expression and isolation, edited by Anne Skaja Robinson
Instantiates
Publication
Bibliography note
Includes bibliographical references and index
Contents
  • Bacterial Systems
  • 2.4.3.
  • CFTR
  • 2.5.
  • Conclusions
  • Abbreviations
  • References
  • 3.
  • Expression Systems: Pichia pastoris
  • Renaud Wagner
  • 3.1.
  • James Samuelson
  • Introduction
  • 3.2.
  • (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
  • 3.4.1.
  • Methanol Utilization Pathway
  • 3.4.2.
  • 1.1.
  • Host Strains and Plasmids
  • 3.4.3.
  • Transformation and Clone Selection Strategies
  • 3.4.4.
  • Expression Conditions and Culturing Formats
  • 3.5.
  • Successful Large-Scale Expression of Membrane Proteins Using P. pastoris
  • 3.5.1.
  • P. pastoris for Membrane Protein Expression
  • 3.5.2.
  • Introduction
  • Common Trends for an Efficient Expression of Membrane Proteins in P. pastoris
  • 3.6.
  • Guidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as Models
  • 3.6.1.
  • Design and Selection of Enhanced Expression Clones
  • 3.6.2.
  • Optimization of the Expression Conditions
  • 3.6.3.
  • Yeast Cell Lysis
  • 3.7.
  • 1.2.
  • Conclusions and Future Directions
  • Acknowledgments
  • Abbreviations
  • References
  • 4.
  • Heterologous Production of Active Mammalian G-Protein-Coupled Receptors Using Baculovirus-Infected Insect Cells
  • Ana Pereda-Lopez
  • 4.1.
  • Introduction
  • 4.2.
  • Understanding the Problem
  • Experimental
  • 4.2.1.
  • Generation of Recombinant Baculovirus
  • 4.2.2.
  • Baculovirus Infection of Insect Cells
  • 4.2.3.
  • Case Study: Histamine H3 Receptor
  • 4.2.3.1.
  • Solubilization of the Histamine H3 Receptor
  • 4.2.3.2.
  • 1.3.
  • Assay Validation
  • 4.2.3.3.
  • Competition Analysis of Solubilized versus Membrane-Bound Receptor
  • 4.3.
  • Conclusions and Future Perspectives
  • 4.3.1.
  • Executive Summary
  • 4.3.2.
  • Future Perspectives
  • Abbreviation
  • Vector/Promoter Types
  • References
  • 5.
  • Membrane Protein Expression in Mammalian Cells
  • Michael J. Betenbaugh
  • 5.1.
  • Introduction
  • 5.2.
  • Mammalian Systems
  • 5.2.1.
  • Cell Culture Types and Media Optimization
  • 1.4.
  • 5.2.1.1.
  • Adherent Cell Culture
  • 5.2.1.2.
  • Suspension Cell Culture
  • 5.2.1.3.
  • Batch and Fed-Batch Culture
  • 5.2.1.4.
  • Perfusion Process
  • 5.2.1.5.
  • Media Optimization
  • T7 Expression System
  • 5.2.2.
  • Gene Delivery and Expression in Mammalian Systems
  • 5.2.2.1.
  • High Transfection Efficiency in Adherent Cell Cultures with Cationic Liposome
  • 5.2.3.
  • Post-Translational Modifications in Mammalian Systems
  • 5.2.3.1.
  • Glycosylation
  • 5.2.3.2.
  • Protein Lipidation
  • Machine generated contents note:
  • 1.5.
  • 5.3.
  • Case Studies
  • 5.3.1.
  • Increasing Membrane Protein Expression by Virus Vectors
  • 5.3.2.
  • Anti-apoptosis Engineering for Increasing Membrane Protein Expression
  • 5.3.3.
  • Increasing Membrane Protein Expression by Chaperones
  • 5.3.4.
  • Membrane Protein Expression in Cancer Cell Lines
  • Tunable T7 Expression Systems
  • 5.3.5.
  • Membrane Proteins as Biotherapeutics
  • 5.4.
  • Conclusions
  • Abbreviations
  • References
  • 6.
  • Membrane Protein Production Using Photosynthetic Bacteria: A Practical Guide
  • Deborah K. Hanson
  • 6.1.
  • 1.6.
  • Introduction
  • 6.1.1.
  • Membrane Protein Problem
  • 6.1.2.
  • Exploiting the Physiology of Photosynthetic Bacteria
  • 6.1.3.
  • Expression Strategies
  • 6.1.3.1.
  • Design of the Expression Plasmids
  • 6.1.3.2.
  • Other Useful Membrane Protein Expression Strains
  • Design of Expression Hosts
  • 6.1.3.3.
  • Autoinduction Conditions
  • 6.1.4.
  • Summary of Success Stories
  • 6.2.
  • Preparation of Expression Constructs
  • 6.2.1.
  • Platform Vector Preparation
  • 6.2.1.1.
  • 1.7.
  • Large-Scale Vector Preparation Protocol for Ligation-Dependent Cloning
  • 6.2.1.2.
  • Large-Scale Vector Preparation Protocol for LIC
  • 6.2.2.
  • Design of Oligonucleotide Primers for Gene Amplification and Cloning
  • 6.2.2.1.
  • Ligation-Dependent Cloning
  • 6.2.2.2.
  • LIC
  • 6.2.3.
  • Clone Stability
  • Target Gene Preparation
  • 6.2.3.1.
  • PCR Amplification of Target Gene
  • 6.2.3.2.
  • Restriction Enzyme Digestion of PCR Amplicon
  • 6.2.3.3.
  • Digestion of PCR Amplicon to Generate LIC Overhangs
  • 6.2.4.
  • Cloning of Digested Amplicons
  • 6.2.4.1.
  • 1.8.
  • Generation of Recombinant Plasmids
  • 6.2.4.2.
  • Transformation of E. coli with Ligation or LIC Reactions
  • 6.2.5.
  • Screening for Successful Insertion of Target Gene into Platform Vector
  • 6.3.
  • Transfer of Plasmid DNA to Rhodobacter via Conjugal Mating
  • 6.3.1.
  • Transformation of E. coli S17-1
  • 6.3.2.
  • Media Types
  • Conjugation of E. coli with R. sphaeroides
  • 6.4.
  • Small-Scale Screening for Expression and Localization of Target Protein in Rhodobacter
  • 6.4.1.
  • Small-Scale Growth and Preparation of Samples for SDS-PAGE
  • 6.4.1.1.
  • Growth and Harvest of Expression Strains
  • 6.4.1.2.
  • Preparing Whole-Cell Samples for SDS-PAGE
  • 6.4.1.3.
  • 1.9.
  • Preparing Membranes and the Soluble Fraction for SDS-PAGE
  • 6.4.2.
  • SDS-PAGE Followed by Electroblotting of Proteins to PVDF Membrane
  • 6.4.3.
  • Immunoblot Development
  • 6.5.
  • Large-Scale Culture
  • 6.5.1.
  • Growth and Harvest of Expression Culture
  • 6.5.2.
  • Fusion Partners/Membrane Targeting Peptides
  • Cell Lysis
  • 6.5.3.
  • Membrane Isolation
  • 6.6.
  • Detergent Solubilization and Chromatographic Purification of Expressed Membrane Proteins
  • 6.6.1.
  • Solubilization of Membrane Proteins
  • 6.6.2.
  • Chromatography
  • 6.6.2.1.
  • Expression
  • 1.10.
  • Bench-Top Affinity Chromatography
  • 6.6.2.2.
  • Affinity Chromatography Using an AKTA-FPLC[TM ]
  • 6.7.
  • Protein Identification and Assessment of Purity
  • 6.8.
  • Preparations of Specialized Rhodobacter Membranes
  • Appendix: Media and Buffer Formulations
  • Abbreviations
  • References
  • Chaperone Overexpression
  • pt. Two
  • Protein-Specific 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
  • 7.2.1.
  • 1.11.
  • Electrostatic Interactions
  • 7.2.1.1.
  • Case 1: Cytochrome c2
  • 7.2.1.2.
  • Case 2: Group IB Secreted Phospholipase A2
  • 7.2.2.
  • Hydrophobic Patch
  • 7.2.2.1.
  • Case 1: Plasma Platelet-Activating Factor Acetylhydrolase
  • 7.2.2.2.
  • Cautionary Notes Related to Chaperone Overexpression
  • Case 2: Human Serum Paraoxonase 1
  • 7.2.3.
  • Covalent Lipid Anchor
  • 7.2.3.1.
  • Case 1: Recoverin
  • 7.2.3.2.
  • Case 2: Intracellular Platelet-Activating Factor Acetylhydrolase Type II
  • 7.2.4.
  • Case 3: Palmitoylation of Human Proteins in Cell Culture
  • 7.2.5.
  • 1.12.
  • Lipid-Binding Domain
  • 7.2.5.1.
  • Case 1: Pleckstrin Homology Domain
  • 7.2.5.2.
  • Case 2: C2 Domain
  • 7.3.
  • Conclusions
  • Acknowledgments
  • Abbreviations
  • References
  • Emerging Role of Quality Control Proteases
  • 8.
  • Expression of G-Protein-Coupled Receptors
  • Krishna Vukoti
  • 8.1.
  • Introduction
  • 8.2.
  • Bacterial Expression of GPCRs
  • 8.3.
  • Expression of GPCRs in Inclusion Bodies, and Refolding
  • 8.4.
  • 1.13.
  • 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.
  • Tag Selection
  • Stabilization of GPCRs during Solubilization and Purification
  • 8.10.
  • Conclusions
  • Acknowledgments
  • Abbreviations
  • References
  • 9.
  • Structural Biology of Membrane Proteins
  • Krzysztof Palczewski
  • 9.1.
  • 1.14.
  • Introduction
  • 9.2.
  • Folding and Structural Analysis of Membrane Proteins
  • 9.2.1.
  • Folding
  • 9.2.2.
  • Prediction Methods
  • 9.2.3.
  • Membrane Insertion
  • 9.2.4.
  • Potential Expression Yield
  • Estimating the Molecular Weight of Membrane Proteins
  • 9.2.5.
  • Amino Acid Composition
  • 9.2.6.
  • Transmembrane Helix Association Motifs and Membrane Protein Oligomerization
  • 9.2.7.
  • Post-Translational Modifications
  • 9.2.7.1.
  • Glycosylation
  • 9.2.7.2.
  • Solubilization and Structural Methods
  • 1.15.
  • Palmitoylation
  • 9.2.8.
  • Sequence Modifications
  • 9.2.9.
  • Lipids and Water
  • 9.2.10.
  • Purity and Contaminants
  • 9.2.11.
  • Current Trends in the Crystallization of α-Helical Membrane Proteins
  • 9.3.
  • Strategies to Overcome Protein Instability
  • Test Cases
  • 9.3.1.
  • Rhodopsin
  • 9.3.2.
  • RPE65
  • 9.3.2.1.
  • Expression in E. coli
  • 9.3.2.2.
  • Expression in Sf9 Cells
  • 9.3.2.3.
  • Acknowledgments
  • Purification from Native Sources
  • 9.3.3.
  • Transmembrane Domain of M2 Protein from Influenza A Virus
  • Acknowledgments
  • Abbreviations
  • References
  • pt. Three
  • Emerging Methods and Approaches --
  • Abbreviations
  • References
  • 2.
  • Membrane Protein Expression in Saccharomyces cerevisiae
  • Anne Skaja Robinson
  • 2.1.
  • Introduction
  • Abbreviations
  • 2.2.
  • Getting Started
  • 2.2.1.
  • Promoter Systems
  • 2.2.1.1.
  • Constitutive Promoters
  • 2.2.1.2.
  • Inducible Promoters
  • 2.2.2.
  • Host Strains, Selection Strategies, and Plasmids
  • References
  • 2.2.2.1.
  • Host Strains
  • 2.2.2.2.
  • Selection Strategies
  • 2.2.2.3.
  • Plasmids and Homologous Recombination
  • 2.2.3.
  • Expression Conditions
  • 2.3.
  • Special Considerations
  • pt. One
  • 2.3.1.
  • Post-Translational Modifications
  • 2.3.1.1.
  • Glycosylation
  • 2.3.1.2.
  • Disulfide Bond Formation
  • 2.3.2.
  • Lipid Requirements
  • 2.3.2.1.
  • Glycerophospholipids
  • Expression Systems
  • 2.3.2.2.
  • Sphingolipids
  • 2.3.2.3.
  • Sterols
  • 2.3.3.
  • Signal Sequences
  • 2.3.4.
  • Topology
  • 2.3.5.
  • Cellular Responses to Membrane Protein Expression
  • 1.
  • 2.3.5.1.
  • UPR
  • 2.3.5.2.
  • HSR
  • 2.4.
  • Case Studies
  • 2.4.1.
  • Ste2p
  • 2.4.2.
  • Pmalp
  • 10.2.1.
  • Affinity
  • 12.3.3.
  • New Approaches and Advances in Purification
  • 12.3.3.1.
  • Magnetic Beads
  • 12.3.3.2.
  • Phase Separation Methods
  • 12.4.
  • Characterization of Solubilized IMPs
  • 12.4.1.
  • Directed Evolution of a GPCR for Higher Expression
  • Sample Homogeneity and Protein Oligomeric State
  • 12.4.1.1.
  • SEC
  • 12.4.1.2.
  • Static Light Scattering (SLS)
  • 12.4.1.3.
  • Analytical Ultracentrifugation (AUC)
  • 12.4.1.4.
  • Blue-Native Electrophoresis (BN-PAGE)
  • 12.4.2.
  • 10.2.2.
  • Structural Characterization
  • 12.4.2.1.
  • Circular Dichroism (CD)
  • 12.4.2.2.
  • IR Spectroscopy
  • 12.4.2.3.
  • NMR Spectroscopy
  • 12.4.3.
  • Measurement and Characterization of Ligand Binding
  • 12.4.3.1.
  • Increasing Expression by Random Mutagenesis and Dot-Blot Based Screening
  • Isothermal Titration Calorimetry (ITC)
  • 12.4.3.2.
  • Spectroscopic Methods
  • Appendix
  • Acknowledgments
  • Abbreviations
  • References
  • 13.
  • Stabilizing Membrane Proteins in Detergent and Lipid Systems
  • Rebecca Batchelor
  • 10.3.
  • 13.1.
  • Introduction
  • 13.2.
  • Choice of Detergent: Solubilization versus Stability
  • 13.2.1.
  • Detergents: General Characteristics
  • 13.2.1.1.
  • Ionic Detergents
  • 13.2.1.2.
  • Zwitterionic Detergents
  • Engineering Higher Stability
  • 13.2.1.3.
  • Nonionic Detergents
  • 13.2.1.4.
  • Detergent-Like Phospholipids
  • 13.2.2.
  • Solubilization
  • 13.3.
  • Mitigating Protein Denaturation
  • 13.3.1.
  • Mixed Detergent Systems
  • 10.3.1.
  • 13.3.1.1.
  • Micelles
  • 13.3.1.2.
  • Bicelles
  • 13.3.2.
  • Detergent-Free Bilayer Systems
  • 13.3.2.1.
  • Lipid Nanodisk
  • 13.3.2.2.
  • Liposomes
  • Stabilizing a Prokaryotic IMP by Cysteine-Scanning, Random Mutagenesis, and Screening in a 96-Well Assay Format
  • 13.3.3.
  • Detergent-Mediated Reconstitution of Proteoliposomes
  • 13.3.3.1.
  • Dilution Method
  • 13.3.3.2.
  • Dialysis versus Hydrophobic Absorption
  • 13.3.3.3.
  • Detergent Saturation
  • 13.3.4.
  • Lipid Composition
  • 10.3.2.
  • 13.3.4.1.
  • Hydrophobic Mismatch
  • 13.3.4.2.
  • Curvature Elastic Stress
  • 13.3.4.3.
  • Specific Lipid Effects
  • 13.4.
  • Making or Selecting a Stable Protein
  • 13.5.
  • Conclusions
  • Stabilizing GPCRs by Alanine-Scanning and Single-Clone Screening
  • Abbreviations
  • References
  • 14.
  • Rapid Optimization of Membrane Protein Production Using Green Fluorescent Protein-Fusions and Lemo21(DE3)
  • Jan-Willem de Gier
  • 14.1.
  • Introduction
  • 14.2.
  • Main Protocol
  • 14.2.1.
  • Contents note continued:
  • 10.3.3.
  • Determination of Membrane Protein Topology and Selection of Expression Vector
  • 14.2.2.
  • Identification of the Optimal Expression Conditions in Lemo21(DE3) Using Whole-Cell and In-Gel Fluorescence
  • 14.2.3.
  • Scaling Up of Expression and Isolation of Membranes
  • 14.2.4.
  • Identification of a Suitable Detergent Using Fluorescence-Detection Size-Exclusion Chromatography
  • 14.2.5.
  • Purification of the Membrane Protein GFP-Fusion and Recovery of the Membrane Protein from the Fusion
  • 14.3.
  • Stabilizing GPCRs by Random Mutagenesis and Screening in a 96-Well Assay Format
  • Materials
  • 14.3.1.
  • Reagents
  • 14.3.2.
  • Equipment
  • 14.4.
  • Expression and Isolation of GFP-His8
  • 14.5.
  • Conclusions
  • Acknowledgments
  • 10.4.
  • Abbreviations
  • References
  • Conclusions
  • Abbreviations
  • References
  • 11.
  • Expression and Purification of G-Protein-Coupled Receptors for Nuclear Magnetic Resonance Structural Studies
  • Sang Ho Park
  • 11.1.
  • 10.
  • 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.
  • Engineering Integral Membrane Proteins for Expression and Stability
  • Cloning of CXCR1 into pGEX2a
  • 11.7.
  • Expression of CXCR1
  • 11.8.
  • Purification
  • 11.9.
  • Refolding and Reconstitution
  • 11.10.
  • Binding and Activity Measurements
  • 11.10.1.
  • Andreas Pluckthun
  • NMR Samples
  • 11.11.
  • NMR Spectra
  • Acknowledgments
  • Abbreviations
  • References
  • 12.
  • Solubilization, Purification, and Characterization of Integral Membrane Proteins
  • Tzvetana Lazarova
  • 12.1.
  • 10.1.
  • Introduction
  • 12.2.
  • Solubilization of IMPs
  • 12.2.1.
  • Physicochemical Characteristics of Detergents
  • 12.2.2.
  • Classification of Detergents
  • 12.2.2.1.
  • Nonionic Detergents
  • 12.2.2.2.
  • Introduction
  • Ionic Detergents
  • 12.2.2.3.
  • Zwitterionic Detergents
  • 12.2.2.4.
  • Recently Developed Detergents
  • 12.2.3.
  • New Solubilizing Agents
  • 12.2.4.
  • Solubilization Process
  • 12.2.5.
  • 10.2.
  • Means of a Successful Solubilization of IMPs
  • 12.2.6.
  • "All" or "Not All" Lipids and If "Purer Is Better"
  • 12.2.7.
  • Stability of the Protein-Detergent Solutions
  • 12.3.
  • IMP Purification
  • 12.3.1.
  • Strategy Definition
  • 12.3.1.1.
  • Engineering Higher Expression
  • HTP Methods
  • 12.3.2.
  • Purification Process
  • 12.3.2.1.
  • Hydrophobicity
  • 12.3.2.2.
  • Charge
  • 12.3.2.3.
  • Size
  • 12.3.2.4.
Dimensions
25 cm.
Extent
xxi, 420 p.
Isbn
9783527634552
Isbn Type
(mobi)
Other physical details
ill.
System control number
  • (CaMWU)u2461457-01umb_inst
  • 2474687
  • (Sirsi) i9783527327294
  • (OCoLC)664325847
Label
Production of membrane proteins : strategies for expression and isolation, edited by Anne Skaja Robinson
Publication
Bibliography note
Includes bibliographical references and index
Contents
  • Bacterial Systems
  • 2.4.3.
  • CFTR
  • 2.5.
  • Conclusions
  • Abbreviations
  • References
  • 3.
  • Expression Systems: Pichia pastoris
  • Renaud Wagner
  • 3.1.
  • James Samuelson
  • Introduction
  • 3.2.
  • (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
  • 3.4.1.
  • Methanol Utilization Pathway
  • 3.4.2.
  • 1.1.
  • Host Strains and Plasmids
  • 3.4.3.
  • Transformation and Clone Selection Strategies
  • 3.4.4.
  • Expression Conditions and Culturing Formats
  • 3.5.
  • Successful Large-Scale Expression of Membrane Proteins Using P. pastoris
  • 3.5.1.
  • P. pastoris for Membrane Protein Expression
  • 3.5.2.
  • Introduction
  • Common Trends for an Efficient Expression of Membrane Proteins in P. pastoris
  • 3.6.
  • Guidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as Models
  • 3.6.1.
  • Design and Selection of Enhanced Expression Clones
  • 3.6.2.
  • Optimization of the Expression Conditions
  • 3.6.3.
  • Yeast Cell Lysis
  • 3.7.
  • 1.2.
  • Conclusions and Future Directions
  • Acknowledgments
  • Abbreviations
  • References
  • 4.
  • Heterologous Production of Active Mammalian G-Protein-Coupled Receptors Using Baculovirus-Infected Insect Cells
  • Ana Pereda-Lopez
  • 4.1.
  • Introduction
  • 4.2.
  • Understanding the Problem
  • Experimental
  • 4.2.1.
  • Generation of Recombinant Baculovirus
  • 4.2.2.
  • Baculovirus Infection of Insect Cells
  • 4.2.3.
  • Case Study: Histamine H3 Receptor
  • 4.2.3.1.
  • Solubilization of the Histamine H3 Receptor
  • 4.2.3.2.
  • 1.3.
  • Assay Validation
  • 4.2.3.3.
  • Competition Analysis of Solubilized versus Membrane-Bound Receptor
  • 4.3.
  • Conclusions and Future Perspectives
  • 4.3.1.
  • Executive Summary
  • 4.3.2.
  • Future Perspectives
  • Abbreviation
  • Vector/Promoter Types
  • References
  • 5.
  • Membrane Protein Expression in Mammalian Cells
  • Michael J. Betenbaugh
  • 5.1.
  • Introduction
  • 5.2.
  • Mammalian Systems
  • 5.2.1.
  • Cell Culture Types and Media Optimization
  • 1.4.
  • 5.2.1.1.
  • Adherent Cell Culture
  • 5.2.1.2.
  • Suspension Cell Culture
  • 5.2.1.3.
  • Batch and Fed-Batch Culture
  • 5.2.1.4.
  • Perfusion Process
  • 5.2.1.5.
  • Media Optimization
  • T7 Expression System
  • 5.2.2.
  • Gene Delivery and Expression in Mammalian Systems
  • 5.2.2.1.
  • High Transfection Efficiency in Adherent Cell Cultures with Cationic Liposome
  • 5.2.3.
  • Post-Translational Modifications in Mammalian Systems
  • 5.2.3.1.
  • Glycosylation
  • 5.2.3.2.
  • Protein Lipidation
  • Machine generated contents note:
  • 1.5.
  • 5.3.
  • Case Studies
  • 5.3.1.
  • Increasing Membrane Protein Expression by Virus Vectors
  • 5.3.2.
  • Anti-apoptosis Engineering for Increasing Membrane Protein Expression
  • 5.3.3.
  • Increasing Membrane Protein Expression by Chaperones
  • 5.3.4.
  • Membrane Protein Expression in Cancer Cell Lines
  • Tunable T7 Expression Systems
  • 5.3.5.
  • Membrane Proteins as Biotherapeutics
  • 5.4.
  • Conclusions
  • Abbreviations
  • References
  • 6.
  • Membrane Protein Production Using Photosynthetic Bacteria: A Practical Guide
  • Deborah K. Hanson
  • 6.1.
  • 1.6.
  • Introduction
  • 6.1.1.
  • Membrane Protein Problem
  • 6.1.2.
  • Exploiting the Physiology of Photosynthetic Bacteria
  • 6.1.3.
  • Expression Strategies
  • 6.1.3.1.
  • Design of the Expression Plasmids
  • 6.1.3.2.
  • Other Useful Membrane Protein Expression Strains
  • Design of Expression Hosts
  • 6.1.3.3.
  • Autoinduction Conditions
  • 6.1.4.
  • Summary of Success Stories
  • 6.2.
  • Preparation of Expression Constructs
  • 6.2.1.
  • Platform Vector Preparation
  • 6.2.1.1.
  • 1.7.
  • Large-Scale Vector Preparation Protocol for Ligation-Dependent Cloning
  • 6.2.1.2.
  • Large-Scale Vector Preparation Protocol for LIC
  • 6.2.2.
  • Design of Oligonucleotide Primers for Gene Amplification and Cloning
  • 6.2.2.1.
  • Ligation-Dependent Cloning
  • 6.2.2.2.
  • LIC
  • 6.2.3.
  • Clone Stability
  • Target Gene Preparation
  • 6.2.3.1.
  • PCR Amplification of Target Gene
  • 6.2.3.2.
  • Restriction Enzyme Digestion of PCR Amplicon
  • 6.2.3.3.
  • Digestion of PCR Amplicon to Generate LIC Overhangs
  • 6.2.4.
  • Cloning of Digested Amplicons
  • 6.2.4.1.
  • 1.8.
  • Generation of Recombinant Plasmids
  • 6.2.4.2.
  • Transformation of E. coli with Ligation or LIC Reactions
  • 6.2.5.
  • Screening for Successful Insertion of Target Gene into Platform Vector
  • 6.3.
  • Transfer of Plasmid DNA to Rhodobacter via Conjugal Mating
  • 6.3.1.
  • Transformation of E. coli S17-1
  • 6.3.2.
  • Media Types
  • Conjugation of E. coli with R. sphaeroides
  • 6.4.
  • Small-Scale Screening for Expression and Localization of Target Protein in Rhodobacter
  • 6.4.1.
  • Small-Scale Growth and Preparation of Samples for SDS-PAGE
  • 6.4.1.1.
  • Growth and Harvest of Expression Strains
  • 6.4.1.2.
  • Preparing Whole-Cell Samples for SDS-PAGE
  • 6.4.1.3.
  • 1.9.
  • Preparing Membranes and the Soluble Fraction for SDS-PAGE
  • 6.4.2.
  • SDS-PAGE Followed by Electroblotting of Proteins to PVDF Membrane
  • 6.4.3.
  • Immunoblot Development
  • 6.5.
  • Large-Scale Culture
  • 6.5.1.
  • Growth and Harvest of Expression Culture
  • 6.5.2.
  • Fusion Partners/Membrane Targeting Peptides
  • Cell Lysis
  • 6.5.3.
  • Membrane Isolation
  • 6.6.
  • Detergent Solubilization and Chromatographic Purification of Expressed Membrane Proteins
  • 6.6.1.
  • Solubilization of Membrane Proteins
  • 6.6.2.
  • Chromatography
  • 6.6.2.1.
  • Expression
  • 1.10.
  • Bench-Top Affinity Chromatography
  • 6.6.2.2.
  • Affinity Chromatography Using an AKTA-FPLC[TM ]
  • 6.7.
  • Protein Identification and Assessment of Purity
  • 6.8.
  • Preparations of Specialized Rhodobacter Membranes
  • Appendix: Media and Buffer Formulations
  • Abbreviations
  • References
  • Chaperone Overexpression
  • pt. Two
  • Protein-Specific 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
  • 7.2.1.
  • 1.11.
  • Electrostatic Interactions
  • 7.2.1.1.
  • Case 1: Cytochrome c2
  • 7.2.1.2.
  • Case 2: Group IB Secreted Phospholipase A2
  • 7.2.2.
  • Hydrophobic Patch
  • 7.2.2.1.
  • Case 1: Plasma Platelet-Activating Factor Acetylhydrolase
  • 7.2.2.2.
  • Cautionary Notes Related to Chaperone Overexpression
  • Case 2: Human Serum Paraoxonase 1
  • 7.2.3.
  • Covalent Lipid Anchor
  • 7.2.3.1.
  • Case 1: Recoverin
  • 7.2.3.2.
  • Case 2: Intracellular Platelet-Activating Factor Acetylhydrolase Type II
  • 7.2.4.
  • Case 3: Palmitoylation of Human Proteins in Cell Culture
  • 7.2.5.
  • 1.12.
  • Lipid-Binding Domain
  • 7.2.5.1.
  • Case 1: Pleckstrin Homology Domain
  • 7.2.5.2.
  • Case 2: C2 Domain
  • 7.3.
  • Conclusions
  • Acknowledgments
  • Abbreviations
  • References
  • Emerging Role of Quality Control Proteases
  • 8.
  • Expression of G-Protein-Coupled Receptors
  • Krishna Vukoti
  • 8.1.
  • Introduction
  • 8.2.
  • Bacterial Expression of GPCRs
  • 8.3.
  • Expression of GPCRs in Inclusion Bodies, and Refolding
  • 8.4.
  • 1.13.
  • 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.
  • Tag Selection
  • Stabilization of GPCRs during Solubilization and Purification
  • 8.10.
  • Conclusions
  • Acknowledgments
  • Abbreviations
  • References
  • 9.
  • Structural Biology of Membrane Proteins
  • Krzysztof Palczewski
  • 9.1.
  • 1.14.
  • Introduction
  • 9.2.
  • Folding and Structural Analysis of Membrane Proteins
  • 9.2.1.
  • Folding
  • 9.2.2.
  • Prediction Methods
  • 9.2.3.
  • Membrane Insertion
  • 9.2.4.
  • Potential Expression Yield
  • Estimating the Molecular Weight of Membrane Proteins
  • 9.2.5.
  • Amino Acid Composition
  • 9.2.6.
  • Transmembrane Helix Association Motifs and Membrane Protein Oligomerization
  • 9.2.7.
  • Post-Translational Modifications
  • 9.2.7.1.
  • Glycosylation
  • 9.2.7.2.
  • Solubilization and Structural Methods
  • 1.15.
  • Palmitoylation
  • 9.2.8.
  • Sequence Modifications
  • 9.2.9.
  • Lipids and Water
  • 9.2.10.
  • Purity and Contaminants
  • 9.2.11.
  • Current Trends in the Crystallization of α-Helical Membrane Proteins
  • 9.3.
  • Strategies to Overcome Protein Instability
  • Test Cases
  • 9.3.1.
  • Rhodopsin
  • 9.3.2.
  • RPE65
  • 9.3.2.1.
  • Expression in E. coli
  • 9.3.2.2.
  • Expression in Sf9 Cells
  • 9.3.2.3.
  • Acknowledgments
  • Purification from Native Sources
  • 9.3.3.
  • Transmembrane Domain of M2 Protein from Influenza A Virus
  • Acknowledgments
  • Abbreviations
  • References
  • pt. Three
  • Emerging Methods and Approaches --
  • Abbreviations
  • References
  • 2.
  • Membrane Protein Expression in Saccharomyces cerevisiae
  • Anne Skaja Robinson
  • 2.1.
  • Introduction
  • Abbreviations
  • 2.2.
  • Getting Started
  • 2.2.1.
  • Promoter Systems
  • 2.2.1.1.
  • Constitutive Promoters
  • 2.2.1.2.
  • Inducible Promoters
  • 2.2.2.
  • Host Strains, Selection Strategies, and Plasmids
  • References
  • 2.2.2.1.
  • Host Strains
  • 2.2.2.2.
  • Selection Strategies
  • 2.2.2.3.
  • Plasmids and Homologous Recombination
  • 2.2.3.
  • Expression Conditions
  • 2.3.
  • Special Considerations
  • pt. One
  • 2.3.1.
  • Post-Translational Modifications
  • 2.3.1.1.
  • Glycosylation
  • 2.3.1.2.
  • Disulfide Bond Formation
  • 2.3.2.
  • Lipid Requirements
  • 2.3.2.1.
  • Glycerophospholipids
  • Expression Systems
  • 2.3.2.2.
  • Sphingolipids
  • 2.3.2.3.
  • Sterols
  • 2.3.3.
  • Signal Sequences
  • 2.3.4.
  • Topology
  • 2.3.5.
  • Cellular Responses to Membrane Protein Expression
  • 1.
  • 2.3.5.1.
  • UPR
  • 2.3.5.2.
  • HSR
  • 2.4.
  • Case Studies
  • 2.4.1.
  • Ste2p
  • 2.4.2.
  • Pmalp
  • 10.2.1.
  • Affinity
  • 12.3.3.
  • New Approaches and Advances in Purification
  • 12.3.3.1.
  • Magnetic Beads
  • 12.3.3.2.
  • Phase Separation Methods
  • 12.4.
  • Characterization of Solubilized IMPs
  • 12.4.1.
  • Directed Evolution of a GPCR for Higher Expression
  • Sample Homogeneity and Protein Oligomeric State
  • 12.4.1.1.
  • SEC
  • 12.4.1.2.
  • Static Light Scattering (SLS)
  • 12.4.1.3.
  • Analytical Ultracentrifugation (AUC)
  • 12.4.1.4.
  • Blue-Native Electrophoresis (BN-PAGE)
  • 12.4.2.
  • 10.2.2.
  • Structural Characterization
  • 12.4.2.1.
  • Circular Dichroism (CD)
  • 12.4.2.2.
  • IR Spectroscopy
  • 12.4.2.3.
  • NMR Spectroscopy
  • 12.4.3.
  • Measurement and Characterization of Ligand Binding
  • 12.4.3.1.
  • Increasing Expression by Random Mutagenesis and Dot-Blot Based Screening
  • Isothermal Titration Calorimetry (ITC)
  • 12.4.3.2.
  • Spectroscopic Methods
  • Appendix
  • Acknowledgments
  • Abbreviations
  • References
  • 13.
  • Stabilizing Membrane Proteins in Detergent and Lipid Systems
  • Rebecca Batchelor
  • 10.3.
  • 13.1.
  • Introduction
  • 13.2.
  • Choice of Detergent: Solubilization versus Stability
  • 13.2.1.
  • Detergents: General Characteristics
  • 13.2.1.1.
  • Ionic Detergents
  • 13.2.1.2.
  • Zwitterionic Detergents
  • Engineering Higher Stability
  • 13.2.1.3.
  • Nonionic Detergents
  • 13.2.1.4.
  • Detergent-Like Phospholipids
  • 13.2.2.
  • Solubilization
  • 13.3.
  • Mitigating Protein Denaturation
  • 13.3.1.
  • Mixed Detergent Systems
  • 10.3.1.
  • 13.3.1.1.
  • Micelles
  • 13.3.1.2.
  • Bicelles
  • 13.3.2.
  • Detergent-Free Bilayer Systems
  • 13.3.2.1.
  • Lipid Nanodisk
  • 13.3.2.2.
  • Liposomes
  • Stabilizing a Prokaryotic IMP by Cysteine-Scanning, Random Mutagenesis, and Screening in a 96-Well Assay Format
  • 13.3.3.
  • Detergent-Mediated Reconstitution of Proteoliposomes
  • 13.3.3.1.
  • Dilution Method
  • 13.3.3.2.
  • Dialysis versus Hydrophobic Absorption
  • 13.3.3.3.
  • Detergent Saturation
  • 13.3.4.
  • Lipid Composition
  • 10.3.2.
  • 13.3.4.1.
  • Hydrophobic Mismatch
  • 13.3.4.2.
  • Curvature Elastic Stress
  • 13.3.4.3.
  • Specific Lipid Effects
  • 13.4.
  • Making or Selecting a Stable Protein
  • 13.5.
  • Conclusions
  • Stabilizing GPCRs by Alanine-Scanning and Single-Clone Screening
  • Abbreviations
  • References
  • 14.
  • Rapid Optimization of Membrane Protein Production Using Green Fluorescent Protein-Fusions and Lemo21(DE3)
  • Jan-Willem de Gier
  • 14.1.
  • Introduction
  • 14.2.
  • Main Protocol
  • 14.2.1.
  • Contents note continued:
  • 10.3.3.
  • Determination of Membrane Protein Topology and Selection of Expression Vector
  • 14.2.2.
  • Identification of the Optimal Expression Conditions in Lemo21(DE3) Using Whole-Cell and In-Gel Fluorescence
  • 14.2.3.
  • Scaling Up of Expression and Isolation of Membranes
  • 14.2.4.
  • Identification of a Suitable Detergent Using Fluorescence-Detection Size-Exclusion Chromatography
  • 14.2.5.
  • Purification of the Membrane Protein GFP-Fusion and Recovery of the Membrane Protein from the Fusion
  • 14.3.
  • Stabilizing GPCRs by Random Mutagenesis and Screening in a 96-Well Assay Format
  • Materials
  • 14.3.1.
  • Reagents
  • 14.3.2.
  • Equipment
  • 14.4.
  • Expression and Isolation of GFP-His8
  • 14.5.
  • Conclusions
  • Acknowledgments
  • 10.4.
  • Abbreviations
  • References
  • Conclusions
  • Abbreviations
  • References
  • 11.
  • Expression and Purification of G-Protein-Coupled Receptors for Nuclear Magnetic Resonance Structural Studies
  • Sang Ho Park
  • 11.1.
  • 10.
  • 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.
  • Engineering Integral Membrane Proteins for Expression and Stability
  • Cloning of CXCR1 into pGEX2a
  • 11.7.
  • Expression of CXCR1
  • 11.8.
  • Purification
  • 11.9.
  • Refolding and Reconstitution
  • 11.10.
  • Binding and Activity Measurements
  • 11.10.1.
  • Andreas Pluckthun
  • NMR Samples
  • 11.11.
  • NMR Spectra
  • Acknowledgments
  • Abbreviations
  • References
  • 12.
  • Solubilization, Purification, and Characterization of Integral Membrane Proteins
  • Tzvetana Lazarova
  • 12.1.
  • 10.1.
  • Introduction
  • 12.2.
  • Solubilization of IMPs
  • 12.2.1.
  • Physicochemical Characteristics of Detergents
  • 12.2.2.
  • Classification of Detergents
  • 12.2.2.1.
  • Nonionic Detergents
  • 12.2.2.2.
  • Introduction
  • Ionic Detergents
  • 12.2.2.3.
  • Zwitterionic Detergents
  • 12.2.2.4.
  • Recently Developed Detergents
  • 12.2.3.
  • New Solubilizing Agents
  • 12.2.4.
  • Solubilization Process
  • 12.2.5.
  • 10.2.
  • Means of a Successful Solubilization of IMPs
  • 12.2.6.
  • "All" or "Not All" Lipids and If "Purer Is Better"
  • 12.2.7.
  • Stability of the Protein-Detergent Solutions
  • 12.3.
  • IMP Purification
  • 12.3.1.
  • Strategy Definition
  • 12.3.1.1.
  • Engineering Higher Expression
  • HTP Methods
  • 12.3.2.
  • Purification Process
  • 12.3.2.1.
  • Hydrophobicity
  • 12.3.2.2.
  • Charge
  • 12.3.2.3.
  • Size
  • 12.3.2.4.
Dimensions
25 cm.
Extent
xxi, 420 p.
Isbn
9783527634552
Isbn Type
(mobi)
Other physical details
ill.
System control number
  • (CaMWU)u2461457-01umb_inst
  • 2474687
  • (Sirsi) i9783527327294
  • (OCoLC)664325847

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