Purification and mass spectroscopic analysis of human CB1 cannabinoid receptor functionally expressed using the baculovirus system.
The cannabinoid receptor 1 (CB1) cannabinoid receptor is an essential component of the cannabinergic system. It has been recognized as a therapeutic target for treating numerous diseases and is currently receiving considerable attention by the pharmaceutical community. Target-based drug design, utilizing three-dimensional information of receptor structure and ligand-binding motifs, requires significant amounts of purified protein. To facilitate the purification of CB1, we have expressed the receptor fused to various epitope tags using the baculovirus expression system. In addition, expression levels and ligand-binding profiles corresponding to the expressed fusion proteins have been compared. C-terminal histidine (His)-tagged CB1 gave a Bmax higher than most other systems previously reported in the literature, and was selected for subsequent metal affinity chromatography purification and mass spectroscopic (MS) analysis. Moreover, cells expressing C-terminal His-tagged CB1 were shown to inhibit forskolin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in a concentration-dependent manner in the presence of CP-55,940, confirming the expressed receptor's functional characteristics. A Western blot analysis of the purified receptor showed several forms of CB1, the most abundant being a 57 kDa monomeric protein. The purified CB1 preparations were subjected to protein digestion followed by MS. Fragments corresponding to >70% of the receptor were identified by this method, confirming the identity and purity of the expressed protein. The work presented here demonstrates that epitope-tagged CB1 can be expressed in sufficient amounts and purified to homogeneity for MS analysis. Moreover, these results will serve as a basis for future experiments aimed at characterizing the ligand-binding domains using covalently reacting receptor probes.
Xu
W
W
Department of Pharmaceutical Sciences, University of Connecticut, 372 Fairfield Road, U-2092, Storrs, CT 06269, USA.
Filppula
S A
SA
Mercier
R
R
Yaddanapudi
S
S
Pavlopoulos
S
S
Cai
J
J
Pierce
W M
WM
Makriyannis
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D043884:Receptor, Cannabinoid, CB1; D011994:Recombinant Proteins