Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants.
Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H(2)L(d) promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.
Reeves
Philip J
PJ
Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Kim
Jong-Myoung
JM
Khorana
H Gobind
HG
D000818:Animals; D019149:Bioreactors; D002417:Cattle; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D011994:Recombinant Proteins; D017299:Rod Opsins; D014415:Tunicamycin; D012243:Rhodopsin; D013752:Tetracycline