Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris.
The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid 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 transformant corresponded to approximately 0.3 mg of opsin per liter of cell culture (A600 = 1.0). Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction. Similar to retinal opsin, P. pastoris-expressed opsin migrated as a single band of approximately 37 kDa on SDS-PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (approximately 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda max 500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment. Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to that of native rhodopsin. These results show that P. pastoris cells have the capacity to functionally express bovine opsin.
Abdulaev
N G
NG
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, Maryland, USA.
Popp
M P
MP
Smith
W C
WC
Ridge
K D
KD
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005796:Genes; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017299:Rod Opsins / Q000096:biosynthesis / Q000235:genetics*
D004271:DNA, Fungal; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D012172:Retinaldehyde