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

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• 作者：Silvia C. Locatelli-Hoops ; Inna Gorshkova ; Klaus Gawrisch ; Alexei A. Yeliseev
• 关键词：CB2 ; peripheral cannabinoid receptor ; GPCR ; G protein-coupled receptor ; MBP ; maltose binding protein ; NTA ; nitrilotriacetic acid ; SPR ; surface plasmon resonance ; TEV ; tobacco etch virus ; CHS ; cholesteryl hemisuccinate Tris salt ; CHAPS ; 3[(cholamidopropyl)
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2013
• 出版时间：October, 2013
• 年：2013
• 卷：1834
• 期：10
• 页码：2045-2056
• 全文大小：823 K

文摘

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.