Multiprotein Complexes: Methods and Protocols

✍ Scribed by Arnaud Poterszman

• Publisher: Humana Press
• Year: 2021
• Tongue: English
• Leaves: 338
• Series: Methods in Molecular Biology, 2247
• Category: Library

✦ Synopsis

This volume explores strategies and detailed protocols for the preparation of macromolecular complexes and their characterization in view of structural analysis. The chapters in this book are separated into three parts: Part One focuses on sample preparation, and covers strategies for recombinant expression of multiprotein complexes in prokaryotic and eukaryotic hosts, for genome engineering using the CRISPR/Cas9 system and for production of specific binders such as reformatted antibodies and artificial binding proteins. Part Two looks at the biophysical methods that can provide useful indicators for sample optimization, and often complement structural information obtained with core technologies for structure determination―x-ray crystallography and cryo-electron microscopy―by quantitative solution data. Part Three discusses the characterization of multiprotein complexes in a cellular environment using the latest technologies and in vivo approaches. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and authoritative, Multiprotein Complexes: Methods and Protocols is a valuable resource for structural and molecular biologists who need to prepare multi-components for their applications, and for other scientists working on macromolecular assemblies from other angles that need to know the latest approaches that the field has to offer.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Recombinant Expression and Isolation from Endogenous Source
Chapter 1: Production of Multi-subunit Membrane Protein Complexes
1 Introduction
1.1 Aim of the Study
1.2 Recombinant Expression in Escherichia coli
1.2.1 Choice of Promoter and Strain
1.2.2 Other Considerations
1.2.3 Recombinant Membrane Protein Expression
1.3 Recombinant Expression in Eukaryotic Expression Systems
1.4 Overexpression in the Endogenous Host
2 Materials
2.1 Expression of HTL
2.2 Membrane Preparation
2.3 Detergent Solubilization
2.4 NiNTA Affinity Chromatography
2.5 Desalting Step
2.6 Calmodulin Affinity Chromatography
2.7 Sample Concentration
2.8 Size Exclusion Chromatography
3 Methods
3.1 Expression of HTL (SecY-SecE-SecG + YidC-SecD-SecF + YajC)
3.2 Membrane Preparation
3.3 Detergent Solubilization
3.4 NiNTA Affinity Chromatography (All Steps at 4 C)
3.5 Desalting Step (at 4 C)
3.6 Calmodulin Affinity Chromatography (at 4 C)
3.7 Sample Concentration
3.8 Size Exclusion Chromatography (Optional)
4 Notes
References
Chapter 2: Production of Multiprotein Complexes Using the Baculovirus Expression System: Homology-Based and Restriction-Free C...
1 Introduction
2 Materials
2.1 Working Environment and Instruments
2.2 Bacterial Strains and Plasmids
2.3 Reagents for the Preparation of the Linearized Vector and DNA Fragments
2.4 Reagents for a SLIC Reaction
2.5 Reagents for an In-Fusion Reaction
2.6 Solution and Media for Restriction-Free Cloning
3 Methods
3.1 Principles of Homology-Based Technologies for Fragment Assembly
3.2 Primer Selection for Homology-Based Cloning
3.3 Preparation of the Linearized Vector and of the DNA Fragments
3.4 SLIC Reaction
3.5 Gibson Assembly, NEBuilder HiFi DNA Assembly, or In-Fusion HD Cloning
3.6 Restriction-Free (RF) Cloning for the Modification of the Existing Constructs
4 Notes
References
Chapter 3: Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors
1 Introduction
2 Materials
2.1 Reagents for the Preparation of sgRNA Expression Plasmids
2.2 Reagents for Nucleofection and Assessment of Gene Editing
2.2.1 Nucleofection with AMAXA Nucleofector Machine
2.2.2 Pool and Clone Analysis
2.3 Reagents for Gene Editing and FACS Sorting
2.3.1 Nucleofection and Antibiotic Selection
2.3.2 FACS Sorting
2.4 Reagents for the Validation of Selected Clones
2.4.1 Genotyping of Single-Cell Clones
2.4.2 Western Blot Analysis
2.4.3 Fluorescence Microscopy
3 Methods
3.1 Design of the sgRNAs
3.2 Delivery of CRISPR Reagents and Assessment of Gene Editing
3.2.1 Nucleofection
3.2.2 PCR-Based Analysis of the Cleavage Efficiency for the sgRNA
3.3 Design of Plasmid Donor
3.4 Gene Targeting and Isolation of Gene Edited Cells by FACS Sorting
3.4.1 Nucleofection and Antibiotic Selection
3.4.2 Isolation of Gene Edited Cells by FACS Sorting
3.5 Validation of Selected Clones
3.5.1 Validation at the Genomic Level
3.5.2 Western Blot Analysis
3.5.3 Fluorescence Microscopy
4 Notes
References
Chapter 4: Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System
1 Introduction
1.1 Aim of the Study
1.2 Expression of Different Recombinant Antibody Formats
1.3 Analysis of the Binding Kinetics of the Antibodies and KLK7 Using Biolayer Interferometry (BLI)
2 Materials
2.1 Expression Vectors
2.2 Cell Lines for Protein Production
2.3 Cell Culture Conditions
2.4 Transfection Reagents and Additional Chemicals for Protein Production
2.5 Biolayer Interferometry
3 Methods
3.1 Transient Protein Expression in HEK293-6E Cells
3.2 Transient Protein Expression in Hi5 Cells
3.3 Baculoviral Expression of hKLK7 in Hi5 Cells
3.4 Preparation of the Baculoviral Stock Solution
3.5 Protein Purification by Affinity Chromatography
3.6 Biolayer Interferometry
4 Notes
References
Chapter 5: The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments
1 Introduction
2 Materials
2.1 Molecular Biology
2.2 Protein Expression Using Brevibacillus Cells
2.3 Protein Expression Using Sf9 Cells and Baculoviruses
2.4 Protein Purification and Characterization
3 Methods
3.1 Original iRAT System for the Production of Recombinant Fv Fragments
3.1.1 Preparation of Brevibacillus Electrocompetent Cells
3.1.2 Construction of the Brevibacillus Expression Plasmid
3.1.3 Secretory Expression Using Brevibacillus Cells
3.1.4 Purification of Tag-Free Fv Fragments
3.2 Extended iRAT System for the Production of Recombinant Fab Fragments and Their Fluorescent Derivatives
3.2.1 Construction of the Baculovirus Strain
3.2.2 Secretory Expression Using Sf9 Insect Cells/Baculoviruses
3.2.3 Purification of Genetically Encoded Fluorescent Fab Fragments
3.2.4 Purification of Tag-Free Fab Fragments
4 Notes
References
Chapter 6: Isolation of Artificial Binding Proteins (Affimer Reagents) for Use in Molecular and Cellular Biology
1 Introduction
2 Materials
2.1 Affimer Selection by Phage Display
2.2 Screening Selected Phage Clones by Phage ELISA
3 Methods
3.1 Affimer Selection by Phage Display
3.1.1 Panning Round 1
3.1.2 Panning Round 2
3.1.3 Panning Round 3
3.2 Screening Selected Phage Clones by Phage ELISA
3.2.1 Expressing Selected Phage Clones in a 96-Well Culture Plate
3.2.2 Phage ELISA
4 Notes
References
Part II: Biophysical Characterization and Sample Optimization
Chapter 7: Measurements of Protein-DNA Complexes Interactions by Isothermal Titration Calorimetry (ITC) and Microscale Thermop...
1 Introduction
2 Materials
2.1 Equipment for Isothermal Titration Calorimetry (ITC)
2.2 Samples for ITC
2.3 Equipment for Microscale Thermophoresis (MST)
2.4 Samples for MST
3 Methods
3.1 Methods for ITC
3.1.1 Experimental Design
3.1.2 Sample Preparation for ITC
3.1.3 ITC Measurements and Data Interpretation
3.2 Methods for MST
3.2.1 Sample Preparation
3.2.2 Fluorescence Pretests
3.2.3 Preliminary Binding Assays
3.2.4 Kd Determination
3.2.5 Assays with Labeled Ku (and Non-labeled DNA)
3.2.6 Assays with Labeled DNA (and Non-labeled Ku)
4 Additional Remarks
References
Chapter 8: switchSENSE Technology for Analysis of DNA Polymerase Kinetics
1 Introduction
2 Materials
2.1 Instrument and Accessories
2.2 Enzyme Preparation
3 Methods
3.1 Polymerase Preparation for switchSENSE Experiment
3.2 Solutions Preparation
3.3 Schematic Overview of the Experiment
3.4 Experimental Design
3.5 Consideration About Obtained Kinetics Parameters
4 Notes
References
Chapter 9: Sedimentation Velocity Methods for the Characterization of Protein Heterogeneity and Protein Affinity Interactions
1 Introduction
1.1 Sedimentation Velocity Equations
1.2 The c(s) Analysis
1.3 Characterization of Protein Heterogeneity Using SV
1.3.1 Non-interacting Versus Interacting System
1.3.2 Non-interacting System Analysis
1.3.3 Example of a Non-interacting Protein Species
1.4 Characterization of Protein Hetero-association Using SV
1.4.1 Lamm Equation Fitting to Characterize Protein Hetero-association
1.4.2 Isotherm Analysis to Characterize Protein Hetero-association
1.4.3 Example of Protein Hetero-association
2 Materials
2.1 Protein Samples
2.2 Instrument and Sample Cells
3 Methods
3.1 Data Acquisition
3.2 Non-interacting System Data Analysis with BSA Data Sets
3.3 Hetero-association Data Analysis with BTG2-PABP Mixtures Data Sets
4 Notes
References
Chapter 10: Hands on Native Mass Spectrometry Analysis of Multi-protein Complexes
1 Introduction
2 Materials
2.1 Desalting
2.2 Cesium Iodide Calibration Mix
2.3 Ammonium Hexafluorophosphate Calibration Mix
2.4 GroEL Reconstitution
3 Methods
3.1 Benchtop Size Exclusion Chromatography Desalting
3.2 Ultrafiltration Desalting
3.3 Direct Infusion of the Sample with the TriVersa Nanomate (Advion)
3.4 Calibration
3.4.1 TOF LCT (Upgraded for High Mass Detection by MS Vision)
3.4.2 Q-TOF Synapt G2 and G2Si
3.4.3 Q-TOF Maxis II
3.4.4 Exactive Plus EMR Orbitrap
3.4.5 Q-Exactive BioPharma Orbitrap
3.5 Native MS Analysis of E. coli RNA Polymerase on a Modified TOF LCT
3.6 Native MS Analysis of the CDK-Activating Kinase Complex on a Q-TOF Synapt G2
3.7 Protein/DNA Complex Analysis on a Q-TOF Synapt G2
3.8 SEC Native MS Coupling (ADH) on a Q-TOF Synapt G2Si
3.9 Homo-Oligomeric Concanavalin A Analysis on a High-Resolution Q-TOF Maxis II
3.10 High Molecular Weight Homo-oligomeric Protein on a High-Resolution Exactive Plus EMR Orbitrap
3.11 Protein Oligomeric State Determination on a High-Resolution Q-Exactive Biopharma Orbitrap
3.11.1 The Trichothiodystrophy Group A Protein (TTD-A or p8)
3.11.2 The Yeast Alcohol Dehydrogenase (ADH)
4 Notes
References
Chapter 11: Studying Protein-DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry
1 Introduction
2 Materials
3 Methods
3.1 Verification of Protein-DNA Complex Formation by a Gel Shift Assay
3.2 Optimizing Digestion Conditions
3.3 Hydrogen/Deuterium Exchange
3.4 Mass Spectrometric Analysis of Deuterated Samples
3.5 Interpreting Data from H/D Exchange
3.5.1 Workflow A: (Largely Automated) Data Interpretation Using DeutEx
3.5.2 Workflow B: Manual Data Interpretation
3.6 Data Visualization
4 Notes
References
Chapter 12: Integrative Mass Spectrometry-Based Approaches for Modeling Macromolecular Assemblies
1 Introduction
1.1 Native MS
1.2 Ion Mobility MS
1.3 Chemical Cross-Linking and Covalent Labelling MS
1.4 Hydrogen-Deuterium Exchange MS
1.5 Integrating with Other Structural Approaches
2 Materials
3 Methods
3.1 Preparing Inputs for IMP
3.2 Sourcing Structures of Constituent Subunits
3.3 Determining Complex Stoichiometry Using Native MS
3.4 Preparation of Cross-Link List for IMP
3.5 Preparation of Stoichiometry List for IMP
3.6 Preparing Modeling Script for IMP
3.7 Modeling Restraints
3.8 Calculating the CCS of Generated Models
4 Notes
References
Chapter 13: Optimization of Sample Preparation for the Observation of Macromolecular Complexes by Electron (cryo-)Microscopy
1 Introduction
1.1 Negative Staining
1.2 Vitrification
2 Materials
2.1 Common Materials and Equipment
2.2 Negative Staining
2.3 Cryo-EM
3 Methods
3.1 Negative Staining
3.1.1 Preparation of a Negatively Stained Sample
3.1.2 Observation and Trouble Shooting
3.2 Vitrification
3.2.1 Preparation of the Vitrified Suspension
3.2.2 Observation and Trouble Shooting
References
Chapter 14: Solubilization and Stabilization of Native Membrane Proteins for Drug Discovery
1 Introduction
2 Materials
2.1 Solubilization Screening
2.2 Radiobinding Assay
2.3 ATPase Activity Assay
2.4 Clear-Native-PAGE
2.5 Glutaraldehyde Cross-Linking Assay
3 Methods
3.1 Screening of Membrane Proteins Solubilization
3.1.1 Cell Lysis and Membrane Fractionation
3.1.2 96-Well Microplate Solubilization
3.1.3 Dot-Blot
3.2 Membrane Protein Stability Assessment
3.2.1 Western Blot-Based Thermostability Assay
3.2.2 Radioligand Binding Assay
3.2.3 ATPase Activity Assay
3.3 Behavior of Membrane Protein in Solution
3.3.1 Clear-Native-PAGE
3.3.2 Glutaraldehyde Cross-Linking Assay
3.3.3 Size Exclusion Chromatography (SEC) Analysis
4 Notes
References
Part III: Analysis in a Cellular Context
Chapter 15: Practical Aspects of Super-Resolution Imaging and Segmentation of Macromolecular Complexes by dSTORM
1 Introduction
2 Materials
2.1 Cell Culture and Immunolabeling
2.2 Mounting Media: Composition and Choice
2.3 Instrumentation
3 Methods
3.1 Specimen Preparation
3.2 Basic SMLM Experiment
3.3 Experimental Parameters of dSTORM Data Collection and Processing
3.4 3D SMLM Experiment
3.5 SMLM Data Processing
3.6 Super-Resolution Image Reconstruction
3.7 Post-processing of Localization Data
3.8 SMLM Data Segmentation and Cluster Analysis, from 2D to 3D
4 Notes
References
Chapter 16: Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in Live Cells
1 Introduction
2 Materials
3 Methods
3.1 Amplification of cDNA by PCR
3.2 Agarose Gel Electrophoresis of PCR Products
3.3 DNA PCR Product Extraction from Agarose and Clean Up
3.4 In-Fusion Reaction with pOPIN Vectors
3.5 Construction of S6K1-mTurq2 and Raptor-YFP Plasmid Constructs
3.6 Cell Lines
3.7 Cell Transfection
3.8 Equipment for Real-Time Imaging of Protein-Protein Interactions Using a Confocal and FRET-FLIM Setup
3.9 Typical Results
4 Notes
References
Chapter 17: Context-Specific and Proximity-Dependent Labeling for the Proteomic Analysis of Spatiotemporally Defined Protein C...
1 Introduction
2 Materials
2.1 Preparation of Plasmids 1
2.2 Transfection of Mammalian Cells
2.3 Proximity-Dependent Biotinylation
2.4 Streptavidin Pulldown
2.5 SDS-Polyacrylamide Gel Electrophoresis and Western Blotting
3 Methods
3.1 Cloning of Two Genes of Interests in Split-BioID Plasmid
3.2 Small-Scale Testing of Proximity-Dependent Biotinylation in Living Cell
3.3 Large-Scale Proximity-Dependent Biotinylation for Proteomics Studies
3.4 Streptavidin Pulldown for Proteomics Studies
3.5 SDS-PAGE and Sample Preparation for MS Analysis
4 Notes
References
Chapter 18: A Directed Evolution System for Lysine Deacetylases
1 Introduction
2 Materials
2.1 Strains and Plasmids (Table 1)
2.2 Creation of KDAC Mutant Libraries by Inverse PCR
2.3 Selection of Lysine Deacetylases
2.4 KDAC Assay Based on Acylated Firefly Luciferase
3 Methods
3.1 Creation of KDAC Mutant Libraries by Inverse PCR
3.1.1 Inverse PCR
3.1.2 Ligation
3.1.3 Electroporation
3.2 Selection of Lysine Deacetylases
3.2.1 Preparation of Electrocompetent Cells (E. coli DeltapyrF DeltacobB pURA3K93TAG-PylS/AcKRS3)
3.2.2 Selection of KDACs
3.2.3 6-AU Assay (Optional)
3.3 KDAC Assay Based on Acylated Firefly Luciferase
3.3.1 Preparation of FLucmod
3.3.2 Purification of CobB
3.3.3 Characterization of Isolated Mutants
4 Notes
References
Index