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SYNTHESIS OF VISUAL RHODOPSIN IN A CELL-FREE TRANSLATION SYSTEM. II. FUNCTIONAL PROPERTIES OF RECOMBINANT RHODOPSIN AND ITS MUTANT FORMS
V. V. GUREVICH, S. A. ZOZULYA, E. P. SHIROKOVA, T. A. ZVYAGA, M. N. GARNOVSKAYA*, I. L. DUMLER*, P. R. BADALOV, M. Yu. NATOCHIN, I. D. POKROVSKAYA, B. E. SHMUKLER
Branch of M. M. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Pushchino , Moscow Region; *I. M. Sechenov Institute of Evolutional Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad
Abstract: Functional expression of bovine visual rhodopsin in the cell-free translation system with cotranslational insertion of the protein into phosphatidylcholine liposomes is described. The recombinant rhodopsin has spectral and functional properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins with amino acid substitutions in the hydrophilic C-terminal domain were obtained using oligonucleotide-directed mutagenesis. It was found that substitution Cys-316→Ser does not affect rhodo-psin's ability to activate the visual amplification cascade, whereas double mutation Asp-330→Asn, Asp-331→Asn dramatically lowers the rhodopsin functional activity.
Russian Journal of Bioorganic Chemistry 1990, 16 (3):303-308