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GPCR Solubilization and Quality Control

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Expression, Purification, and Structural Biology of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2127))

Abstract

G protein-coupled receptors (GPCRs) are versatile membrane proteins involved in the regulation of many physiological processes and pathological conditions, making them interesting pharmacological targets. In order to study their structure and function, GPCRs are traditionally extracted from membranes using detergents. However, due to their hydrophobic nature, intrinsic instability in aqueous solutions, and their denaturing effects, the isolation of properly folded and functional GPCRs is not trivial. Therefore, it is of crucial importance to solubilize receptors under mild conditions and control the sample quality subsequently. Here we describe widely used methods for small-scale GPCR solubilization, followed by quality control based on fluorescence size-exclusion chromatography, SDS-PAGE, temperature-induced protein unfolding (CPM dye binding) and fluorescent ligand binding assay. These methods can easily be used to assess the thermostability and functionality of a GPCR sample exposed to different conditions, such as the use of various detergents, addition of lipids and ligands, making them valuable for obtaining an optimal sample quality for structural and functional studies.

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Author information

Authors and Affiliations

  1. Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland

    Tamara Miljus, Ziva Vuckovic & Dmitry B. Veprintsev

  2. Department of Biology, ETH Zürich, Zürich, Switzerland

    Tamara Miljus, Ziva Vuckovic & Dmitry B. Veprintsev

  3. Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK

    Tamara Miljus, David A. Sykes, Clare R. Harwood & Dmitry B. Veprintsev

  4. Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

    Tamara Miljus

  5. Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK

    David A. Sykes, Clare R. Harwood & Dmitry B. Veprintsev

  6. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia

    Ziva Vuckovic

Authors
  1. Tamara Miljus
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  2. David A. Sykes
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  3. Clare R. Harwood
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  4. Ziva Vuckovic
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  5. Dmitry B. Veprintsev
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Corresponding author

Correspondence to Dmitry B. Veprintsev .

Editor information

Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Camilo Perez

  2. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Timm Maier

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Miljus, T., Sykes, D.A., Harwood, C.R., Vuckovic, Z., Veprintsev, D.B. (2020). GPCR Solubilization and Quality Control. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0373-4_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0372-7

  • Online ISBN: 978-1-0716-0373-4

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