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

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

Klammt, Christian
•
Perrin, Marilyn H.
•
Maslennikov, Innokentiy
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May 3, 2011
Protein Science

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 2 beta 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.

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Type
research article
DOI
10.1002/pro.636
Author(s)
Klammt, Christian
Perrin, Marilyn H.
Maslennikov, Innokentiy
Renault, Ludovic
Krupa, Martin
Kwiatkowski, Witek
Stahlberg, Henning  orcid-logo
Vale, Wylie
Choe, Senyon
Date Issued

2011-05-03

Publisher

Wiley-Blackwell

Published in
Protein Science
Volume

20

Issue

6

Start page

1030

End page

1041

Editorial or Peer reviewed

REVIEWED

Written at

OTHER

EPFL units
LBEM  
Available on Infoscience
February 13, 2020
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/165460
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