A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors
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Author(s)
Baaske, Philipp
Geissler, Sandra
Wienken, Christoph J.
Jerabek-Willemsen, Moran
Duhr, Stefan
Braun, Dieter
Corin, Karolina A.
Ravel, Deepali B.
Song, Junyao
Brown, Emily E.
Date Issued
October 2011
Journal
PLoS ONE
Publisher
Public Library of Science
Citation
Corin, Karolina et al. “A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors.” Ed. Jian R. Lu. PLoS ONE 6.10 (2011): e23036. Web. 10 Feb. 2012.
Version
Final published version
Abstract
Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.
MIT Department
Massachusetts Institute of Technology. Center for Biomedical Engineering
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
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DOI of Published Version
https://doi.org/10.1371/journal.pone.0023036
