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Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents

Klammt, C. and Perrin, M. H. and Maslennikov, I. and Renault, L. and Krupa, M. and Kwiatkowski, W. and Stahlberg, H. and Vale, W. and Choe, S.. (2011) Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents. Protein Science, Vol. 20, H. 6. pp. 1030-1041.

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Official URL: http://edoc.unibas.ch/dok/A5848227

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Abstract

G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2beta are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)
UniBasel Contributors:Stahlberg, Henning
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Cambridge University Press
ISSN:0961-8368
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:14 Sep 2012 07:17
Deposited On:14 Sep 2012 06:42

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