Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents.
Détails
ID Serval
serval:BIB_3D83F131E76A
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents.
Périodique
Protein science
ISSN
1469-896X (Electronic)
ISSN-L
0961-8368
Statut éditorial
Publié
Date de publication
06/2011
Peer-reviewed
Oui
Volume
20
Numéro
6
Pages
1030-1041
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Publication Status: ppublish
Résumé
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 2β 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.
Mots-clé
Cell-Free System/metabolism, Detergents, Fructans/chemistry, Fructans/metabolism, Gene Expression, Humans, Ligands, Nuclear Magnetic Resonance, Biomolecular, Polymers/chemistry, Polymers/metabolism, Protein Binding, Receptors, G-Protein-Coupled/genetics, Receptors, G-Protein-Coupled/metabolism
Pubmed
Web of science
Création de la notice
09/06/2023 15:03
Dernière modification de la notice
28/07/2023 5:59