Data Entry: Please note that the research database will be replaced by UNIverse by the end of October 2023. Please enter your data into the system https://universe-intern.unibas.ch. Thanks

Login for users with Unibas email account...

Login for registered users without Unibas email account...

 
Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
 
ID 832530
Author(s) Klammt, Christian; Perrin, Marilyn H; Maslennikov, Innokentiy; Renault, Ludovic; Krupa, Martin; Kwiatkowski, Witek; Stahlberg, Henning; Vale, Wylie; Choe, Senyon
Author(s) at UniBasel Stahlberg, Henning
Year 2011
Title Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents
Journal Protein Science
Volume 20
Number 6
Pages / Article-Number 1030-41
Keywords cell-free, family B GPCR, functional ligand binding, CRFR, integral membrane protein, polymer, NMR, static light scattering
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.

Publisher Cambridge University Press
ISSN/ISBN 0961-8368
URL http://www.ncbi.nlm.nih.gov/pubmed/21465615
edoc-URL http://edoc.unibas.ch/dok/A5848227
Full Text on edoc No
Digital Object Identifier DOI 10.1002/pro.636
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/21465615
ISI-Number WOS:000291068000009
Document type (ISI) Journal Article
 
   

MCSS v5.8 PRO. 0.404 sec, queries - 0.000 sec ©Universität Basel  |  Impressum   |    
03/05/2024