FUNCTIONAL EXPRESSION OF BOVINE OPSIN IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS

The methylotrophic yeast Pichia pastoris was examined for functional e xpression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alco hol oxidase 1 gene promoter and fused at its amino-terminus to the aci d phosphatase secretion signal, Quantitative-competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome. The expression level in this tra nsformant corresponded to similar to 0.3 mg of opsin per liter of cell culture (A(600) = 1.0). Sucrose density sedimentation analysis indica ted that the opsin was associated exclusively with the membrane fracti on. Similar to retinal opsin, P. pastoris-expressed opsin migrated as a single band of similar to 37 kDa on SDS-PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (similar to 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda( max) 500 nm), and after purification showed ground and excited state s pectral characteristics indistinguishable from those of the native pig ment. Further, the metarhodopsin-II-mediated G-protein-activating pote ntial of yeast expressed rhodopsin was similar to that of native rhodo psin. These results show that P. pastoris cells have the capacity to f unctionally express bovine opsin. (C) 1997 Academic Press.