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Comparative Study
. 1996 Oct 15;93(21):11482-6.
doi: 10.1073/pnas.93.21.11482.

Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae

R Mollaaghababa  1 , F F Davidson, C Kaiser, H G Khorana
Affiliations
Comparative Study

Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae

R Mollaaghababa et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 1997 Apr 1;94(7):3481

Abstract

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.

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