{METHODS} {FOR} {RECOMBINANT} {EXPRESSION} {AND} {FUNCTIONAL} {CHARACTERIZATION} {OF} {HUMAN} {CANNABINOID} {RECEPTOR} {CB2}

摘要

Cannabinoid receptor {CB2} is a seven transmembrane-domain integral membrane protein that belongs to a large superfamily of G protein-coupled receptors (GPCR). {CB2} is a part of the endocannabinoid system that plays vital role in regulation of immune response, inflammation, pain sensitivity, obesity and other physiological responses. Information about the structure and mechanisms of functioning of this receptor in cell membranes is essential for the rational development of specific pharmaceuticals. Here we review the methodology for recombinant expression, purification, stabilization and biochemical characterization of {CB2} suitable for preparation of multi-milligram quantities of functionally active receptor. The biotechnological protocols include expression of the recombinant {CB2} in E. coli cells as a fusion with the maltose binding protein, stabilization with a high affinity ligand and a derivative of cholesterol in detergent micelles, efficient purification by tandem affinity chromatography, and reconstitution of the receptor into lipid bilayers. The purified recombinant {CB2} receptor is amenable to functional and structural studies including nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.