title: Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system.
abstract: The cannabinergic system is present in a variety of organs and tissues that perform a wide range of 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) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane-bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)-terminal His-tagged CB2 construct displayed a B(max) value of 9.3 pmol/mg with a K(D) of 7.30 nM using [3(H)]CP-55(940), a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high-level expression systems when developing membrane-bound protein purification strategies. This work will aid in the identification of receptor-ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.
authors.affiliation: Center for Drug Discovery, Department of Pharmaceutical Sciences and Department of Molecular and Cell Biology, University of Connecticut, 372 Fairfield Road, U-2092, Storrs, CT 06269, USA.
authors.forename: S
authors.initials: S
authors.lastname: Filppula
authors.forename: S
authors.initials: S
authors.lastname: Yaddanapudi
authors.forename: R
authors.initials: R
authors.lastname: Mercier
authors.forename: W
authors.initials: W
authors.lastname: Xu
authors.forename: S
authors.initials: S
authors.lastname: Pavlopoulos
authors.forename: A
authors.initials: A
authors.lastname: Makriyannis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D043885:Receptor, Cannabinoid, CB2; D011994:Recombinant Proteins