Functional expression in vitro of bovine visual rhodopsin

Abstract

Expression of functionally active bovine visual rhodopsin was achieved by sequential transcription and translation in vitro of rhodopsin gene cDNA with co-translational insertion of the protein into phosphatidylcholine liposomes. The recombinant rhodopsin has functional, spectral and immunochemical properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins, Cys316 .fwdarw. Ser and Asp330 .fwdarw. Asn, Asp331 .fwdarw. Asn, were produced by oligonucleotide-directed mutagenesis. The first mutation does not affect rhodopsin''s ability to stimulate transducin GTPase and visual cGMP phosphodiesterase activities, while the second double mutation leads to a sharp decrease in rhodopsin activity.