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This book collects up-to-date advanced protocols and advice from leading experts in the area of membrane protein biology that can be applied to structural and functional studies of any membrane protein system. The contents explore methods for cloning and expression of membrane proteins and membrane protein complexes in prokaryotic and eukaryotic systems, approaches for protein purification, nanobody applications, as well as biophysical characterization and much more. Written for the highly successful Methods in Molecular Biology series, 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.
Authoritative and thorough, Expression, Purification, and Structure Biology of Membrane Proteins serves to guide and encourage young researchers and newcomers to the field to tackle bold new studies on membrane proteins.
Chapter 11 is available open access under a CC-BY 4.0 license via link.springer.com.
List of contents
Cloning and Multi-Subunit Expression of Mitochondrial Membrane Protein Complexes in Saccharomyces cerevisiae.- Membrane Protein Production in Escherichia coli.- Membrane Protein Production in Lactococcus Lactis for Structural Studies.- Expression and Purification of Membrane Proteins in Saccharomyces cerevisiae.- Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.- Membrane Extracts from Plant Tissues.- Membrane Protein Solubilization and Quality Control: An Example of a Primary Active Transporter.- GPCR Solubilization and Quality Control.- Affinity Purification of Membrane Proteins.- Purification of Membrane Proteins by Affinity Chromatography with On-Column Protease Cleavage.- Biotinylation of Membrane Proteins for Binder Selections.- Production and Application of Nanobodies for Membrane Protein Structural Biology.- Identifying Conformation-Selective Heavy-Chain-Only Antibodies against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.- Reconstitution of Membrane Proteins into Platforms Suitable for Biophysical and Structural Analyses.- Reconstitution of Proteoliposomes for Phospholipid Scrambling and Non-Selective Channel Assays.- Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent.- Single-Particle Cryo-EM of Membrane Proteins in Lipid Nanodiscs.- Fast Small-Scale Membrane Protein Purification and Grids Preparation for Single Particle Electron Microscopy.- Stabilization and Crystallization of a Membrane Protein Involved in Lipids Transport.- In Meso In Situ Serial X-ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source.- Membrane Protein Preparation for Serial Crystallography Using High Viscosity Injectors: Rhodopsin as an Example.- Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins.- Mechanical Unfolding and Refolding of Single Membrane Proteins by Atomic Force Microscopy.- Sample Preparation and Technical Setup for NMR Spectroscopy with Integral Membrane Proteins.- Exploring Lipid- and Membrane Protein-Dynamics Using Lipid-Bilayer Nanodiscs and Solution-State NMR Spectroscopy.
