Heterologous Expression of Membrane Proteins: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Solubilization and Purification of α5β1 Integrin from Rat Liver for Reconstitution into Nanodiscs
1 Introduction
2 Materials
2.1 General Materials
2.2 α5β1 Integrin Solubilization
2.3 α5β1 Integrin Purification and Characterization
2.4 α5β1 Integrin Reconstitution into Nanodiscs
2.5 α5β1 Integrin Characterization in Both Micelles and Nanodisc-Reconstituted Formats, Using Electron Microscopy (EM)
3 Methods
3.1 Preparation of Rat Livers Lysates (Solubilization)
3.2 Purification of Glycosylated Proteins on a Wheat-Germ Agglutinin (WGA) Column
3.3 Purification of α5β1 Integrin on a FnIII9-10 Fibronectin Fragment Column
3.4 Characterization of α5β1 Integrin Purity by SDS-PAGE
3.5 Estimation of α5β1 Integrin Protein Concentration
3.6 Assessment of α5β1 Integrin Heterodimer Identity
3.7 Reconstitution of α5β1 Integrin in Nanodiscs (ND)
3.7.1 Lipid Mixture Preparation
3.7.2 Reconstitution of α5β1 Integrin into Nanodiscs
3.8 Purification of Nanodiscs Containing α5β1 Integrin
3.9 EM Characterization of α5β1 Integrin/Micelles and α5β1 Integrin/ND
4 Notes
References
Chapter 2: Membrane Protein Production in Escherichia coli: Protocols and Rules
1 Introduction
2 Materials
2.1 Materials
2.1.1 For RNA Isolation
2.1.2 For Sucrose Gradient and Solubilization of Membranes
2.1.3 For Flow Cytometry
2.1.4 For Membrane or Inclusion Body Collection
2.1.5 For Fluorescence Microscopy
2.2 Media and Chemicals
2.3 Web Resources
2.3.1 Sequence Analysis and Molecular Biology Tools
2.3.2 Vector Design
2.3.3 Molecular and Structural Biology Websites
3 Methods
3.1 Designing Constructs for Expression
3.2 Selecting the Optimal Expression Vector/Bacterial Host
3.3 Viability Test on Agar Plate
3.4 Selecting a Host Strain Adapted to the Expression of Your Target MP
3.4.1 Selection Procedure
3.4.2 Localization of the Mutation
3.5 Optimization of Growth Conditions
3.5.1 Exploring Induction Conditions and Mutant Hosts by Flow Cytometry
3.5.2 Testing the mRNA Stability: Phenol-Chloroform RNA Extraction from E. coli
3.6 Collecting Membranes or Inclusion Bodies from E. coli
3.6.1 Check the Presence of Inclusion Bodies
3.6.2 Collecting E. coli Membranes in the Absence of Inclusion Bodies
3.6.3 Sucrose Gradient Protocol
3.6.4 Testing the Solubility of Your Target MP
3.7 Labelling E. coli Membranes by Flow Cytometry and Fluorescence Microscopy
3.7.1 Flow Cytometry Analysis
3.7.2 Fluorescence Microscopy
4 Notes
References
Chapter 3: Functional Overexpression of Membrane Proteins in E. coli: The Good, the Bad, and the Ugly
1 Introduction
2 Materials
2.1 Growth Media
2.2 Chemicals, Reagents, and Equipment
2.3 Buffers Used for the Multidrug ABC Transporter BmrA (See Note 1)
3 Methods
3.1 Expression Vectors
3.2 E. Coli Strains and Induction Conditions
3.2.1 Selection of E. coli Strains
3.2.2 Combination with Useful Plasmids
3.2.3 Induction Conditions
3.3 Overexpression of Membrane Proteins: General Guidelines
3.4 Preparation of the Membrane Fraction
3.5 Quality Assessment of Membrane Protein Expression
3.5.1 GFP-Fusion Based Systems
3.5.2 Functional Characterization of Membrane Proteins
3.5.3 Solubilization of Membrane Proteins by Mild Detergents
3.6 Case Studies
3.6.1 BmrA, a Multidrug ABC Transporter from Bacillus subtilis
3.6.2 A BceAB-Type Transporter from Streptococcus pneumoniae
3.7 Expression of Soluble Proteins
4 Notes
References
Chapter 4: Heterologous Expression of Membrane Proteins in E. coli
1 Introduction
2 Materials
2.1 Molecular Cloning
2.1.1 Polymerase Chain Reaction (PCR)
2.1.2 Restriction Digestion
2.1.3 Ligation
2.1.4 Heat-Shock Transformation
2.2 Small Scale Expression
2.3 SDS-PAGE and Western Blotting
2.4 Cloning and Expression of a Toxic Outer Membrane Protein
3 Methods
3.1 Molecular Cloning
3.1.1 Polymerase Chain Reaction (PCR)
3.1.2 Restriction Digestion
3.1.3 Ligation
3.1.4 Heat-Shock Transformation
3.1.5 Verification of Clones by Diagnostic Digestion
3.2 Small Scale Expression of a Recombinant Protein at Different Temperatures
3.3 SDS-PAGE and Western Blotting
3.4 Cloning and Expression of a Toxic Outer Membrane Protein
4 Notes
References
Chapter 5: Overexpression of the ABC Transporter BmrA Within Intracellular Caveolae in Escherichia coli
1 Introduction
2 Materials
2.1 Plasmids
2.2 Lab Equipments
2.3 SDS-PAGE
2.4 Cells and Media for Cell Culture
2.5 Membrane Preparation and Sucrose Gradient
2.6 ATPase Activity
2.7 Hoechst 33342 Transport Assays
3 Methods
3.1 E. coli Cotransformation with Genes Coding for BmrA and MBP-Cav1β
3.2 Culture and Overexpression
3.3 Membrane Preparation
3.4 Probe BmrA Expression on SDS-PAGE, Quantify Expression Using Prepurified Protein
3.5 ATPase Activity
3.6 Sucrose Gradient and SDS-PAGE Analysis
3.7 Hoechst 33342 Transport Assays (Fig. 4)
4 Notes
References
Chapter 6: Production of Membrane Proteins in Pseudomonas stutzeri
1 Introduction
2 Materials
2.1 Strains
2.2 Media and Stock Solutions
2.3 Construction of the Expression Vector
2.4 Electroporation of P. stutzeri Cells
2.5 Small-Scale Protein Expression Analysis
2.6 Membrane Preparation and Solubilization
2.7 Protein Purification
2.8 Equipment and Instruments
3 Methods
3.1 Cloning of the Gene of Interest into Pseudomonas Expression Vector
3.1.1 Selection of the Vectors
3.1.2 Selection of the Cloning Strategies
3.1.3 Cloning via LIC
3.1.4 Cloning via Digestion and Ligation
3.1.5 Transformation into E. coli
3.1.6 Isolation of Plasmid DNA
3.1.7 Insertion Check by Digestion or Sequencing
3.2 Electroporation of Expression Vectors into P. stutzeri
3.2.1 Preparation of P. stutzeri Electrocompetent Cells
3.2.2 Electrotransformation of P. stutzeri (See Note 9)
3.2.3 Verification of the Recombinant Vector
3.3 Expression of the GOI in P. stutzeri
3.3.1 Cultivation of P. stutzeri
3.3.2 Storage of P. stutzeri
3.4 Small-Scale Protein Expression Analysis
3.4.1 Small-Scale Expression Screening
3.4.2 Analysis of Protein Expression by SDS-PAGE and Western Blot
3.4.3 Analysis of Protein Expression by Dot-Blot Analysis
3.4.4 GFP Folding Assay
3.5 Large-Scale Protein Expression and Purification
3.5.1 Membrane Preparation
3.5.2 Solubilization
3.5.3 Affinity Chromatography
3.5.4 Optional: TEV-Protease Cleavage
3.5.5 Size-Exclusion Chromatography
3.5.6 Biochemical Analysis of Purified Proteins
4 Notes
References
Chapter 7: Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae
1 Introduction
2 Materials
2.1 Cloning Target MIP/MAP Gene into Yeast Expression Vector
2.2 Preparing Competent S. cerevisiae Cells
2.3 Transforming Competent S. cerevisiae Cells
2.4 S. cerevisiae Growth and Protein Expression
2.5 Protein Expression Validation via Immunodetection and Fluorescent Screening
2.6 Required Equipment
3 Methods
3.1 Cloning Target Gene(s) into Yeast Expression Vector(s)
3.2 Generating Competent S. cerevisiae Cells
3.3 Transforming S. cerevisiae Cells with Expression Plasmid
3.4 Overexpressing MAPs/MIPs in S. cerevisiae Cells
3.5 Protein Preparation, Immunodetection, and Fluorescent Screening
3.6 Optimizing Membrane Protein Overexpression in S. cerevisiae
4 Notes
References
Chapter 8: Purification of Membrane Proteins Overexpressed in Saccharomyces cerevisiae
1 Introduction
2 Materials
2.1 Solubilization Screening and Expression Characterization
2.2 Immobilized Metal Affinity Purification of Membrane Proteins
2.3 Size Exclusion Chromatography (SEC) Purification
2.4 Ion Exchange Chromatography Purification
3 Methods
3.1 Solubilization Screening and Expression Characterization
3.2 IMAC MP Purification Using a Chromatography System
3.3 Modifications for IMAC Purification Without a Chromatography System
3.4 Size-Exclusion Chromatography
3.5 Ion Exchange Chromatography
4 Notes
References
Chapter 9: Production and Purification of a GFP-Tagged ABC Transporter CaCdr1p
1 Introduction
2 Materials
2.1 Lab Equipment
2.2 Yeast Cell Culture and Overexpression
2.3 Bacterial Culture and Overexpression
2.4 Nanobody Purification
2.5 Yeast Lysis and Membrane Isolation
2.6 Detergent Screening and Protein Purification
3 Methods
3.1 Yeast Cell Culture and Overexpression
3.2 Bacterial Culture and Overexpression
3.3 Nanobody Purification
3.4 Yeast Lysis and Membrane Isolation
3.5 Detergent Screening and Protein Purification
3.6 Example of a Purification Procedure Using trans-PCC α-M
4 Notes
References
Chapter 10: Membrane Protein Production in the Yeast P. pastoris
Abbreviations
1 Introduction
2 Materials
2.1 Stock Solutions for P. pastoris Growth
2.2 P. pastoris Strain and Culture Media
2.3 Equipment for Recombinant CD81 Production in Shake Flasks
2.4 Equipment for Recombinant CD81 Production in Bioreactors
3 Methods
3.1 Expression of Recombinant CD81 Using Shake Flasks
3.1.1 Small Scale Growth
3.1.2 Large-Scale Growth
3.2 Expression of Recombinant CD81 Using a Bioreactor
4 Notes
References
Chapter 11: Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Ana...
1 Introduction
2 Materials
2.1 MP Production in Labeling Conditions
2.2 Yeast Cell Lysis and Membrane Preparation
2.3 Extraction of MPs from Membrane Preparation
2.4 Purification of Solubilized MPs
2.4.1 Immobilized Affinity Chromatography (IMAC)
2.4.2 Size Exclusion Chromatography (SEC)
2.5 SDS-PAGE and Western Blot Immunodetection
2.5.1 SDS-PAGE
2.5.2 Western Blotting and Immunodetection
2.6 Reconstitution in MLVs
2.7 Sample Packing in Fast-MAS Rotors
3 Methods
3.1 Production of MPs in Isotope-Labeling Conditions with Pichia pastoris
3.2 Yeast Cell Lysis and Whole Membrane Preparation
3.3 Extraction of MPs from Membrane Preparation
3.4 Purification of Solubilized MPs
3.4.1 IMAC
3.4.2 SEC
3.5 SDS-PAGE and Western Blot Immunodetection
3.5.1 10% SDS Tricine Polyacrylamide Gel Preparation
3.5.2 Gel Electrophoresis and Coomassie Staining
3.5.3 Protein Transfer and Western Blot Immunodetection
3.6 Reconstitution in MLVs
3.7 Sample Packing in Fast-MAS NMR Rotors
4 Notes
References
Chapter 12: Membrane Protein Production in Insect Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 General Cell Culture Materials
2.3 Cell Freezing
2.4 P0 Virus Stock Production
2.4.1 Bac-to-Bac
2.4.2 flashBAC
2.5 Protein Production
3 Methods
3.1 Establishment and Maintenance of a Suspension Cell Culture
3.1.1 Cell Thawing
3.1.2 Monolayer Cell Passage
3.1.3 Transferring Cells to a Shaker Flask
3.1.4 Maintaining a Stock of Cells in Shaker Flasks
3.1.5 Cell Freezing
3.2 Virus Production and Amplification
3.2.1 P0 Virus Production Using the Bac-to-Bac System (Based on the Bac-to-Bac Handbook)
3.2.2 P0 Virus Production Using the flashBAC System
3.2.3 P0 to P1 Virus Amplification
3.2.4 P1 to P2 (or Higher) Virus Amplification
3.3 Recombinant Protein Production
3.3.1 Small Scale Test Expression
3.3.2 Large Scale Expression
3.3.3 Membrane Preparation
4 Notes
References
Chapter 13: Thromboxane A2 G Protein-Coupled Receptor Production and Crystallization for Structure Studies
1 Introduction
2 Materials
2.1 Bacmid DNA Generation
2.2 High-Titer Recombinant Baculovirus Generation
2.3 TPR Large-Scale Expression
2.4 TPR Purification
2.5 TPR In Meso Crystallization
3 Methods
3.1 Bacmid DNA Generation
3.2 High-Titer Recombinant Baculovirus Generation
3.3 TPR Large-Scale Expression
3.4 TPR Purification
3.5 TPR In Meso Crystallization
4 Notes
References
Chapter 14: Production of Human ABC Transporters and Oligosaccharyltransferase Complexes for Structural Studies
1 Introduction
2 Materials
2.1 Membrane Protein Expression
2.2 Protein Purification
2.2.1 Chemicals and Materials
2.2.2 Purification Buffers
2.3 Analysis of the Sample Quality and Functional Assays
2.3.1 ATPase Assays for ABC Transporters
2.3.2 Antibodies for Western Blots
2.3.3 In-Vitro Glycosylation Assays for OST
3 Methods
3.1 Generation of Stable Cell Lines
3.2 Expression and Purification of Human ABCB1
3.2.1 Expression
3.2.2 Purification
3.2.3 Nanodisc Reconstitution
3.2.4 Quality Analysis: ATPase Activity
3.3 Expression and Purification of Human OST Complexes
3.3.1 Expression
3.3.2 Purification in Detergent
3.3.3 Quality Analysis: Western Blot Analysis
3.3.4 Quality Analysis: In-Vitro Glycosylation Assays
3.4 Cryo-EM Analysis of Human ABCB1 and OST Complexes
4 Notes
References
Chapter 15: Heterologous Expression and Purification of GPCRs
1 Introduction
2 Materials
2.1 Plasmids and Recombinant Bacmid Generation
2.2 Reagents and Buffers for Insect Cell Culture and Baculovirus Infection
2.3 Reagents and Buffers for Mammalian Cell Culture
2.4 Cell Membrane Preparation and Solubilization
2.5 Purification of Solubilized GPCRs
2.6 Lab Apparatus
3 Methods
3.1 Plasmid Preparation
3.2 Recombinant Bacmid Preparation
3.3 Expression of GPCRs in Insect Cells
3.3.1 Initiate Insect Cell Culture from Frozen Stock
3.3.2 Cell Maintenance
3.3.3 Cell Counting
3.3.4 Virus Production
3.3.5 Virus Titration
3.3.6 Virus Amplification
3.3.7 Small-Scale Expression
3.3.8 Large-Scale Expression
3.4 Expression of GPCRs in Mammalian Cells
3.5 Cell Pellets Lysis and Membrane Solubilization
3.5.1 40 ml Biomass Membrane Washing Steps
3.5.2 1 l Biomass Membrane Washing Steps
3.5.3 A Small-Scale Biomass (40 ml) Membrane Solubilization Steps
3.5.4 A Large-Scale Biomass (1 l) Membrane Solubilization Steps
3.6 Purification of the Protein
3.6.1 A Small-Scale (40 ml Biomass) Purification
3.6.2 A Large-Scale (1 l Biomass) Purification
3.7 Co-Expression and Purification of GPCR-G Protein Complexes
4 Notes
References
Chapter 16: Expression and Purification of the Human Thyroid-Stimulating Hormone Receptor
1 Introduction
2 Materials
2.1 Generation of a Stable Cell Line and Receptor Expression
2.2 Receptor Purification
3 Methods
3.1 Generation of a Stable Cell Line: Lentivirus Production and Transduction
3.2 Generation of a Stable Cell Line: FACS and Cell Line Expansion
3.3 Expression of TSHR
3.4 Purification of TSHR: Preparation of Whole Cell Lysate
3.5 Purification of TSHR: IMAC
3.6 Purification of TSHR: FLAG Immunoaffinity Purification
3.7 Quality Control
4 Notes
References
Chapter 17: Optimization of Recombinant GPCR Proteins for Biophysical and Structural Studies Using Virus-like Particles
1 Introduction
2 Materials
2.1 pCMV-GAG Plasmid Generation
2.2 pEF6-GPCR Plasmid Generation
2.3 Cell Culture and Transfection
2.4 GalR3 Radioligand Binding Assay
2.5 MS-Based Thermal Stability Assay
3 Methods
3.1 pCMV-GAG Plasmid Generation
3.2 pEF6-GPCR Plasmid Generation
3.3 Transfection and VLP Production
3.4 VLP Harvest
3.5 GPCR Radioligand Binding Assay Development, GalR3 as an Example
3.6 GPCR Yield and Thermal Stability Measurement by Radioligand Binding Assay, GalR3 as an Example (See Note 8)
3.7 GPCR Thermal Stability Assay by LC/MS, GalR3 as an Example (See Note 10, Fig. 3)
4 Notes
References
Chapter 18: Peptide Tags and Domains for Expression and Detection of Mammalian Membrane Proteins at the Cell Surface
1 Introduction
1.1 Peptide Tags and Domains
1.2 Mammalian Expression Vectors and Cloning
1.3 Transient Transfection
1.4 SLC Family
1.5 Transport Assay
2 Materials
2.1 Equipment
2.2 Cloning of the Target Gene
2.3 Tissue Culture Consumables
2.4 Transfection and Adherent Cell Culture
2.5 Expression of Target Protein
2.6 Cellular Localization of Target Protein
2.7 Transport Assays with Whole Cells
3 Methods
3.1 Construction of an Expression Plasmid Containing the Recombinant Gene with Different Epitope Tags and Domains
3.2 Transient Transfection of Target Protein in HEK293FT Cells
3.3 Cell Harvest and Detection of the Recombinant Protein
3.4 Protein Detection and Localization Using Confocal Microscopy
3.5 Transport Assays with Whole Cells Expressing Different Constructs
4 Notes
References
Chapter 19: Tailoring Purification and Analysis of Membrane Proteins with Modular Detergents
1 Introduction
2 Materials
2.1 Membrane Protein Expression and Membrane Preparation
2.2 Oligoglycerol Detergent Stock Solutions
2.3 Extraction and IMAC Purification
2.4 CD Spectroscopy
2.5 Native Mass Spectrometry
3 Methods
3.1 Membrane Protein Expression and Membrane Preparation
3.2 Detergent Selection
3.3 Membrane Protein Extraction and IMAC Purification
3.4 CD Spectroscopy of Membrane Proteins
3.5 Native Mass Spectrometry of Membrane Proteins
4 Notes
References
Chapter 20: Detergent Alternatives: Membrane Protein Purification Using Synthetic Nanodisc Polymers
1 Introduction
2 Materials
2.1 Membrane Isolation and Solubilization
2.2 Protein Purification and Quality Control
3 Methods
3.1 Membrane Isolation
3.2 Membrane Solubilization Screen
3.3 Membrane Protein Purification with the SMALP 200 Polymer
4 Notes
References
Chapter 21: Detergent-Free Membrane Protein Purification Using SMA Polymer
1 Introduction
2 Materials
2.1 SMA Polymer Preparation
2.2 Membrane Protein Extraction and Purification
3 Methods
3.1 SMA Polymer Preparation
3.2 Membrane Protein Extraction and Purification
4 Notes
References
Chapter 22: Cell-Free Expression of GPCRs into Nanomembranes for Functional and Structural Studies
1 Introduction
2 Materials
2.1 General Laboratory Equipment
2.2 Preparation of Stocks for CF Protein Expression
2.3 Preparation of E. coli S30 Lysate
2.4 Expression and Purification of T7 RNAP
2.5 Expression and Purification of Membrane Scaffold Protein
2.6 Nanodisc Formation
2.7 Continuous-Exchange Cell-Free Protein Expression
2.8 Radioligand Binding Assay
2.9 Ligand Affinity Chromatography (LAC)
3 Methods
3.1 Preparation of E. coli S30 Lysate
3.1.1 Standard S30 Lysate
3.1.2 Chaperone-Enriched HS30 Heat Shock Lysate
3.2 Expression and Purification of T7 RNAP
3.3 Preforming Empty Nanodiscs
3.3.1 Expression and Purification of Membrane Scaffold Protein
3.3.2 Nanodisc Assembly
3.4 Continuous Exchange Cell-Free Protein Synthesis
3.5 Functional Optimization of Cell-Free GPCR Expression
3.5.1 Optimizing Redox Conditions and Lysate Composition for CF GPCR Expression
3.5.2 Identification of Suitable Membrane Lipid Compositions
3.5.3 Radioligand Binding Assay
3.6 Ligand Affinity Chromatography of Cell-Free Expressed GPCRs for Structural Approaches
4 Notes
References
Chapter 23: Cell-Free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs
1 Introduction
1.1 Structure and Function of Membrane Protein Depends on Lipid Bilayers
1.2 Proton-Couple Folate Transporter
1.3 Cell-Free Expression Versus Traditional Expression for Membrane Proteins
1.4 Introduction of PCFT-pEXP5-NT Construct
1.5 Introduction of Cell-Free Expression System
1.6 Introduction of Nanodiscs
2 Materials
2.1 Equipment and Software
2.2 Reagents and Chemicals
3 Methods
3.1 Designing and Cloning PCFT-pEXP5-NT Construct
3.2 Cell-Free Expression of PCF-PCFT Protein Synthesis
3.3 Immunoblot Analysis-Western Blot
3.3.1 SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis)
3.3.2 Transferring Proteins from Gel to PVDF Membrane
3.3.3 Block Available Binding Sites on PVDF Membranes
3.3.4 Chemiluminescent Detection of PCFT Cell-Free Expression Protein
3.4 Protein Quantification Using ImageJ Software
3.5 Statistics and Figures
4 Notes
References
Chapter 24: Microscale Thermophoresis to Evaluate the Functionality of Heterologously Overexpressed Membrane Proteins in Membr...
1 Introduction
2 Materials
2.1 Strains
2.2 Vectors Preparation
2.3 Yeast Transformation
2.4 Yeast Culture
2.5 Yeast Membrane Fraction Preparation
2.6 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.7 Western Blot
2.8 Microscale Thermophoresis
3 Methods
3.1 Construction of Expression Vectors
3.2 Yeast Transformation
3.3 Yeast Culture for Expression of Your Target MDR Protein
3.4 Membrane Preparation
3.5 Protein Expression Analysis by Western Blotting
3.6 Binding Affinity Measure on Membranes from Ptch1 Expressing Yeast Using Microscale Thermophoresis
3.6.1 Determine the Affinity of the His-Labeling Dye RED-Tris-NTA for the Target Protein and to Set the Membrane Concentration...
3.6.2 Protein Labeling and Binding Affinity Assay
3.7 Examples on Other Membrane Proteins
3.7.1 Using a GFP Labeled Target Instead of a His-Tag Labeling
3.7.2 Stochastic Labeling on Total Membrane Extract
4 Notes
References
Index