Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae.
The yeast Saccharomyces cerevisiae has been investigated for expression of mammalian opsin as an alternative to the currently used expression in COS-1 mammalian cells. The synthetic opsin gene was placed under the control of the inducible promoter GAL1 in the multicopy yeast/ Escherichia coli shuttle vector YEpRF1. Transformation of a GAL+ S. cerevisiae strain with the vector and growth of galactose-induced cultures to saturation showed the production of 2.0 +/- 0.5 mg of opsin from about 10(10) cells by ELISA. The addition of 11-cis-retinal to either cell spheroplasts or lysed cells showed that a fraction (2-4%) of the total expressed opsin reconstituted to rhodopsin. This fraction was purified to homogeneity and was shown to be fully functional and indistinguishable from bovine rhodopsin by the following criteria: (i) UV-visible absorption spectra, (ii) the formation of metarhodopsin II and its rate of decay, and (iii) initial rate of transducin activation as measured by the formation of a complex between transducin (alpha subunit) and guanosine 5'-[gamma-[35S]thio]triphosphate. The purified fraction was homogeneously glycosylated. However, glycosylation was distinct from that of bovine rhodopsin as judged by mobility on SDS/PAGE and endoglycosidase H sensitivity.
Mollaaghababa
R
R
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Davidson
F F
FF
Kaiser
C
C
Khorana
H G
HG
D000818:Animals; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D011401:Promoter Regions, Genetic; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D017299:Rod Opsins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013053:Spectrophotometry; D013104:Spheroplasts; D015293:Transducin / Q000378:metabolism
D017299:Rod Opsins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde