title: Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1.
abstract: G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the beta1 and beta2 adrenergic receptors and the A2A adenosine receptor. This lag in obtaining GPCR structures is due to several tedious steps that are required before beginning the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. With the aim to obtain active membrane receptors for functional and crystallization studies, we recently reported a screen of expression conditions for approximately 100 GPCRs in Escherichia coli, providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step. Here, we report a novel artificial chaperone-assisted refolding procedure adapted for the GPCR inclusion body refolding, followed by protein purification and characterization. The refolding of two selected targets, the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid hormone receptor 1 (huPTH1R), was achieved from solubilized receptors using detergent and cyclodextrin as protein folding assistants. We could demonstrate excellent affinity of both refolded and purified receptors for their respective ligands. In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS, and Universités of Marseille, F-13288 Marseille Cedex 09, France.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Michalke
authors.forename: Céline
authors.initials: C
authors.lastname: Huyghe
authors.forename: Julie
authors.initials: J
authors.lastname: Lichière
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Gravière
authors.forename: Marina
authors.initials: M
authors.lastname: Siponen
authors.forename: Giuliano
authors.initials: G
authors.lastname: Sciara
authors.forename: Isabelle
authors.initials: I
authors.lastname: Lepaul
authors.forename: Renaud
authors.initials: R
authors.lastname: Wagner
authors.forename: Christine
authors.initials: C
authors.lastname: Magg
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Aline
authors.initials: A
authors.lastname: Desmyter
mesh_terms: D000818:Animals; D003505:Cyclodextrins / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017510:Protein Folding; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003505:Cyclodextrins; D043884:Receptor, Cannabinoid, CB1; D044168:Receptor, Parathyroid Hormone, Type 1; D011994:Recombinant Proteins