A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors
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%...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Public Library of Science
2012
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| Online Access: | http://hdl.handle.net/1721.1/69080 |
| _version_ | 1811094435646144512 |
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| author | 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. Wang, Xiaoqiang Zhang, Shuguang |
| author2 | Massachusetts Institute of Technology. Center for Biomedical Engineering |
| author_facet | Massachusetts Institute of Technology. Center for Biomedical Engineering 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. Wang, Xiaoqiang Zhang, Shuguang |
| author_sort | Baaske, Philipp |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T16:00:00Z |
| format | Article |
| id | mit-1721.1/69080 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T16:00:00Z |
| publishDate | 2012 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/690802022-10-02T05:39:13Z A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors 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. Wang, Xiaoqiang Zhang, Shuguang Massachusetts Institute of Technology. Center for Biomedical Engineering Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Zhang, Shuguang Corin, Karolina A. Ravel, Deepali B. Song, Junyao Brown, Emily E. Wang, Xiaoqiang Zhang, Shuguang 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. United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012) Massachusetts Institute of Technology. Undergraduate Research Opportunities Program 2012-02-10T17:28:07Z 2012-02-10T17:28:07Z 2011-10 2011-04 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/69080 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. en_US http://dx.doi.org/10.1371/journal.pone.0023036 PLoS ONE Creative Commons Attribution http://creativecommons.org/licenses/by/2.5/ application/pdf Public Library of Science PLoS |
| spellingShingle | 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. Wang, Xiaoqiang Zhang, Shuguang A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors |
| title | A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors |
| title_full | A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors |
| title_fullStr | A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors |
| title_full_unstemmed | A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors |
| title_short | A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors |
| title_sort | robust and rapid method of producing soluble stable and functional g protein coupled receptors |
| url | http://hdl.handle.net/1721.1/69080 |
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