Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants
Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants
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DOI：
10.1073/pnas.212519199
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发表时间：
2002-10-15
影响因子：
11.1
通讯作者：
Khorana, HG
中科院分区：
文献类型：
--
作者：
Reeves, PJ;Kim, JM;Khorana, HG
已制备出四环素诱导的HEK293S稳定细胞系，仅在四环素和丁酸钠的加入下表达高水平（高达10 mg/l）的WT视蛋白及其突变体。用含有tetO序列的巨细胞病毒启动子下游的视蛋白基因和由弱H2Ld启动子控制的新霉素耐药基因的表达质粒稳定转染hek292s - tetr细胞制备细胞系。该诱导系统特别适合于克服由于在生长培养基中添加有毒化合物（例如，tunicamycin）或由于表达的蛋白产物而引起的毒性问题。通过优化细胞在生物反应器中的生长条件，WT视蛋白（一种组成活性视蛋白突变体E113Q/E134Q/M257Y）被认为对细胞有毒性，而在tunicamycin存在下生长获得的非糖基化WT视蛋白的量为每升培养基中几毫克。
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 H2Ld 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.