High-level inducible expression of visual pigments in transfected cells.
A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.
Kazmi
M A
MA
New York University Medical Center, NY, USA.
Dubin
R A
RA
Oddoux
C
C
Ostrer
H
H
D000818:Animals; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012172:Retinaldehyde / Q000737:chemistry; D012243:Rhodopsin / Q000096:biosynthesis* / Q000235:genetics; D017299:Rod Opsins / Q000096:biosynthesis* / Q000235:genetics; D012680:Sensitivity and Specificity; D015293:Transducin / Q000378:metabolism; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D001616:beta-Galactosidase; D015293:Transducin; D019256:Cadmium Chloride; D012172:Retinaldehyde