Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2

• Published in: Biochimica et biophysica acta 2013-10, Vol.1834 (10), p.2045-2056
• Main Authors: Locatelli-Hoops, Silvia C., Gorshkova, Inna, Gawrisch, Klaus, Yeliseev, Alexei A.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Cannabinoid receptor CB2; Detergents - chemistry; Escherichia coli; Escherichia coli - genetics; Functional immobilization; Gene Expression; Humans; Immobilized Proteins - chemistry; Immobilized Proteins - genetics; Kinetics; Ligands; Micelles; Protein Array Analysis; Receptor, Cannabinoid, CB2 - chemistry; Receptor, Cannabinoid, CB2 - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Rho-tag; Rhodopsin - chemistry; Rhodopsin - genetics; Surface plasmon resonance (SPR); Thermodynamics
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.06.003

Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2. •Expressed and purified functional recombinant cannabinoid receptor CB2•Immobilized CB2 in oriented fashion onto antibody-coated Biacore CM4 chip•Characterized functional recombinant CB2 by ligand binding in detergent micelles•Characterized binding of Rho-tagged CB2 to surface-immobilized 1D4 antibody by SPR•Characterized binding of monoclonal antibody to surface-immobilized CB2