Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system

摘要: The cannabinergic system is present in a variety of organs and tissues that perform wide range essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) their ligands, efficient effective tools are robust expression purification these membrane-bound proteins. this report, we describe suitable method human receptor 2 (CB2) qualitative quantitative level sufficient mass spectral analysis. We utilized baculovirus system, incorporating several epitope tags facilitate ameliorate effect have on CB2 function. Expressed protein encoded by carboxy (C)-terminal His-tagged construct displayed B(max) value 9.3 pmol/mg with K(D) 7.30 nM using [3(H)]CP-55(940), standard radioligand, was selected subsequent experiments. Western blot analysis purified membrane yielded forms CB2, most abundant being 41 kDa peptide. A second species observed apparent molecular weight 46 representing glycosylated form CB2. addition, homodimer also identified. subjected spectroscopic confirm its identity purity. Mass spectra corresponding intracellular, extracellular transmembrane domains were obtained. These experiments exemplify importance high-level systems when developing strategies. This work will aid identification receptor-ligand binding sites, characterization features involved activation, elucidation tertiary structure.