title: Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2.
abstract: 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.
authors.affiliation: National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, Bethesda, MD 20892, USA.
authors.forename: Silvia C
authors.initials: SC
authors.lastname: Locatelli-Hoops
authors.forename: Inna
authors.initials: I
authors.lastname: Gorshkova
authors.forename: Klaus
authors.initials: K
authors.lastname: Gawrisch
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Yeliseev
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008823:Micelles; D040081:Protein Array Analysis; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D003902:Detergents; D055767:Immobilized Proteins; D008024:Ligands; D008823:Micelles; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin
keywords: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; 3[(cholamidopropyl)dimethylammonio]-1-propanesulfonate; CB(2); CHAPS; CHS; Cannabinoid receptor CB(2); DDM; DTPA; Functional immobilization; G protein-coupled receptor; GPCR; MBP; N-octyl-β-d-glucopyranoside; NTA; OG; POPC; POPS; Rho-tag; SPR; Surface plasmon resonance (SPR); TEV; cholesteryl hemisuccinate Tris salt; diethylenetriamine pentaacetate; maltose binding protein; n-dodecyl-β-d-maltoside; nitrilotriacetic acid; peripheral cannabinoid receptor; surface plasmon resonance; tobacco etch virus