Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris—Importance of correct processing of the N-terminus

Abstract

This study was conducted to optimize the expression of human CB2 cannabinoid receptors in methylotrophic yeast Pichia pastoris ( P. pastoris). Two major species of expressed CB2 proteins were seen on Western blot, i.e., a 42 kDa band which matches the calculated molecular weight for tagged CB2, and a 52/55 kDa doublet. Treatment of membranes with N-glycosidase F or inclusion of tunicamycin in the culture medium during induction resulted in the disappearance of the 55 kDa, but not the 52 kDa band, suggesting that the 3 kDa extra in the 55 kDa band is due to N-glycosylation, but the 10 kDa extra in the 52 kDa band is not due to N-glycosylation. Anti-FLAG M1 antibody had a much higher preference for the 42 kDa band over the 52/55 kDa doublet, and a 10 kDa fragment recognized by anti-FLAG M2 antibody was generated by CNBr digestion of the 52/55 doublet. These data strongly support the hypothesis that the 10 kDa increase in molecular weight was due to unprocessed α-factor sequence. This conclusion was further validated by finding several peptide sequences for α-factor fragments at the N-terminal of the CB2 receptor using pepsin/chymotrypsin digestion and LC/MS/MS approaches. Importantly, unprocessed α-factor was found to be associated with poor ligand binding. In addition, controlling the level of CB2 protein expression was found to be critical for minimizing the presence of unprocessed α-factor sequence. The information gained from this study should aid the proper expression of not only CB2 receptor but also other members of the GPCR family in P. pastoris.