Mammalian GPCR expression in Escherichia coli. Part 2: Refolding and Biophysical Characterization of muCB1R and huPTH1R

G protein-coupled receptors (GPCRs) represent ~3% of the human proteome. They are involved in a large number of diverse processes and are therefore the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the β 1 and β 2 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 first have recently reported a screen of expression conditions for ~100 GPCRs in E. coli , providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step (Michalke et al . 2009 Anal. Biochem. 386,147-155). 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.