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Bacterial expression and functional and structural characterization of human peripheral cannabinoid receptor CB2

Human peripheral cannabinoid receptor CB2 was expressed in Escherichia coli as a fusion with maltose binding protein, streptag and a decahistidine tag. Functionality of the receptor was confirmed by ligand‐binding studies and by activation of the cognate G proteins in an in vitro coupled assay. Recombinant CB2 was solubilized in a mixture of detergents: CHAPS, dodecylmaltoside (DM) and cholesterol hemisuccinate (CHS), and purified by immobilized metal affinity chromatography. The expression partners were removed by cleaving the fusion with specific TEV protease and the resulting CB2 purified by affinity chromatography on StrepTactin to 85–90% homogeneity. Structural properties and stability of the recombinant CB2 were studied by circular dichroism spectroscopy (CD). The α‐helical content of the recombinant receptor in detergent micelles, as determined by CD, was at least 54%. Partial denaturation of the protein induced by increasing temperature reduced α‐helical content to 30–40%. Replacement, by site‐directed mutagenesis, of six different amino acid residues predicted to be involved in the interaction of CB2 with specific ligands or activation of cognate G proteins generated recombinant cannabinoid receptors with altered functional properties. Mutated forms of the receptor were purified to apparent homogeneity. We are initiating spectroscopic studies of the wild type and mutated receptors in detergent micelles and in proteoliposomes that will provide insight into the mechanisms of ligand binding and activation of CB2.