Multiprotein Complexes Methods and Protocols

Preface…

Table of Contents…

Contributing Authors…

Part I

1. Production of Multi-Subunit Membrane Protein Complexes

Burak V. Kabasakal, Qiyang Jiang, and Christiane Schaffitzel

2. Production of Multiprotein Complexes using the Baculovirus Expression System: Homology Based and Restriction Free Cloning Strategies for Construct Design

Paola Rossollillo, Olga Kolesnikova, Karim Essabri, Gala Ramon Zamorano, and Arnaud Poterszman

3. Tagging Proteins with Fluorescent Reporters using the CRISPR/Cas9 System and Double Stranded DNA Donors

Sylvain Geny, Simon Pichard, Alice Brion, Jean-Baptiste Renaud, Sophie Jacquemin, Jean-Paul Concordet, and Arnaud Poterszman

4. Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System

Jens König, Michael Hust, and Joop van den Heuvel

5. The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments

Norimichi Nomura, Yayoi Nomura, Yumi Sato, and So Iwata

6. Isolation of Artificial Binding Proteins (Affimer Reagents) for Use in Molecular and Cellular Biology

Anna A. S. Tang, Christian Tiede, Michael J. McPherson, and Darren C. Tomlinson

Part II

7. Interactions Measurements of Protein-DNA Complexes by Isothermal Titration Calorimetry (ITC) and Microscale Thermophoresis (MST)

Amandine Gontier, Paloma F. Varela, Clément Nemoz, Virginie Ropars, Magali Aumont-Nicaise, Michel Desmadril, and Jean-Baptiste Charbonnier

8. switchSENSE Technology for Analysis of DNA Polymerase Kinetics

Guillaume Bec and Eric Ennifar 

9. Sedimentation Velocity Methods for Characterization of Protein Heterogeneity and Protein Affinity Interactions

Christine Ebel and Catherine Birck

10. Hands on Native Mass Spectrometry Analysis of Multi-Protein Complexes

Stéphane Erb, Sarah Cianférani, and Julien Marcoux

Ruzena Filandrova, Daniel Kavan, Alan Kadek, Petr Novak, and Petr Man

12. Integrative Mass Spectrometry-Based Approaches for Modeling Macromolecular Assemblies

Andy M. Lau and Argyris Politis

13. Optimization of Sample Preparation for the Observation of Macromolecular Complexes by Electron (cryo-)Microscopy

Alexandre Frechard, Grigory Sharov, Maximilien Werderer, and Patrick Schultz

14. Solubilization and Stabilization of Native Membrane Proteins for Drug Discovery

Corvest Vincent and Jawhari Anass

Part III

15. Practical Aspects of Super-Resolution Imaging and Segmentation of Macromolecular Complexes by dSTORM

Leonid Andronov, Jean-Luc Vonesch, and Bruno P. Klaholz

16. Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in Live Cells

Abdullah Ahmed, Jennifer Schoberer, Emily Cooke, and Stanley W. Botchway

17. Context-Specific and Proximity-Dependent Labeling for the Proteomic Analysis of Spatiotemporally Defined Protein Complexes with Split-BioID

Cinthia Amaya Ramirez, Stefanie Egetemaier, and Julien Béthune

18. A Directed Evolution System for Lysine Deacetylases

Martin Spinck, Maria Ecke, Damian Schiller, and Heinz Neumann

Subject Index List…

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.