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%...

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Autores principales:	Karolina Corin, Philipp Baaske, Deepali B Ravel, Junyao Song, Emily Brown, Xiaoqiang Wang, Sandra Geissler, Christoph J Wienken, Moran Jerabek-Willemsen, Stefan Duhr, Dieter Braun, Shuguang Zhang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/08442d96fbdd466ca1606d4e76038eda
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spelling	oai:doaj.org-article:08442d96fbdd466ca1606d4e76038eda2021-11-18T07:35:50ZA robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.1932-620310.1371/journal.pone.0023036https://doaj.org/article/08442d96fbdd466ca1606d4e76038eda2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22039398/?tool=EBIhttps://doaj.org/toc/1932-6203Membrane 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.Karolina CorinPhilipp BaaskeDeepali B RavelJunyao SongEmily BrownXiaoqiang WangSandra GeisslerChristoph J WienkenMoran Jerabek-WillemsenStefan DuhrDieter BraunShuguang ZhangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 10, p e23036 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Karolina Corin Philipp Baaske Deepali B Ravel Junyao Song Emily Brown Xiaoqiang Wang Sandra Geissler Christoph J Wienken Moran Jerabek-Willemsen Stefan Duhr Dieter Braun Shuguang Zhang A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.
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.
format	article
author	Karolina Corin Philipp Baaske Deepali B Ravel Junyao Song Emily Brown Xiaoqiang Wang Sandra Geissler Christoph J Wienken Moran Jerabek-Willemsen Stefan Duhr Dieter Braun Shuguang Zhang
author_facet	Karolina Corin Philipp Baaske Deepali B Ravel Junyao Song Emily Brown Xiaoqiang Wang Sandra Geissler Christoph J Wienken Moran Jerabek-Willemsen Stefan Duhr Dieter Braun Shuguang Zhang
author_sort	Karolina Corin
title	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_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_sort	robust and rapid method of producing soluble, stable, and functional g-protein coupled receptors.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/08442d96fbdd466ca1606d4e76038eda
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A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

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