Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae with the copper-inducible CUP1 promoter.
Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae with the copper-inducible CUP1 promoter.
复制标题
使用铜诱导型 CUP1 启动子在酿酒酵母中表达酒精诱导型兔肝细胞色素 P-450 3a (P-450IIE1)。
DOI：
10.1089/dna.1990.9.111
复制
发表时间：
1990
影响因子：
3.1
通讯作者：
Coon,MJ
中科院分区：
文献类型：
--
作者：
Fujita,VS;Thiele,DJ;Coon,MJ
利用铜诱导的酵母金属硫蛋白（CUP 1）启动子和ADH 1启动子，在酿酒酵母中表达酒精诱导的兔肝微粒体细胞色素P-450 3a型（P450 IIE 1）的cDNA。比较菌株50.L4和PP 1002的最佳表达蛋白水平。免疫印迹分析表明，细胞色素P-450 3a在菌株50.L4中获得了更高的表达水平，而在两种启动子下，未诱导的表达蛋白水平相似。在CUP 1启动子的作用下，在二价铜离子的存在下，cDNA的转录被强烈诱导，并且在菌株50.L4中表达的免疫反应性蛋白的量增加了20倍，使得其占总细胞蛋白的0.8%。这些细胞的细胞色素P-450全酶含量，从减少CO差谱计算，约为0.02 nmole/mg蛋白质，或总细胞蛋白质的0.1%。从这些细胞制备的微粒体的全酶含量高达0.06 nmole/mg蛋白质，或微粒体蛋白质的0.4%。微粒体测定乙烯形成N-亚硝基二乙胺和苯胺羟基化，两个典型的纯化兔细胞色素P-450形式3a的反应，表明在酵母中合成的细胞色素催化这两个反应。此外，多克隆抗3a IgG完全抑制与酵母微粒体中的两种底物的反应。比较这些底物的产物比率表明，酵母中表达的细胞色素P-450 3a具有与真实兔蛋白相似的催化活性。
The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) inSaccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and theADH1promoter, is described. Strains 50.L4 and PP1002 were compared for optimal levels of expressed protein. Immunoblot analysis showed that a much higher level of expression of cytochrome P-450 3a is obtained with strain 50.L4, and that the uninduced levels of expressed protein are similar with the two promoters. With theCUP1promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed is increased 20-fold in strain 50.L4, such that it constitutes 0.8% of the total cellular protein. The cytochrome P-450 holoenzyme content of these cells, calculated from the reduced CO difference spectrum, is about 0.02 nmole/mg of protein, or 0.1% of the total cellular protein. The holoenzyme content of microsomes prepared from these cells is up to 0.06 nmole/mg of protein, or 0.4% of the microsomal protein. Microsomal assays for ethylene formation fromN-nitrosodiethylamine and for anilinep-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions. Further-more, polyclonal anti-3a IgG completely inhibits the reactions with both substrates in yeast microsomes. A comparison of the product ratios from these substrates showed that the cytochrome P-450 3a expressed in yeast has catalytic activities similar to those of the authentic rabbit protein.
登录
查看更多内容
转化酵母细胞中合成的兔肝细胞色素 P-450（月桂酸 omega-1 羟化酶）的表征。
DOI：
--
发表时间：
1988
期刊：
Journal of Biochemistry (Tokyo)
影响因子：
--
作者：
Y. Imai
通讯作者：
Y. Imai
串联复制的上游控制序列介导铜诱导的酿酒酵母铜金属硫蛋白基因的转录
影响因子：
5.3
作者：
D. Thiele;D. Hamer
通讯作者：
D. Hamer
N-亚硝基乙胺依赖细胞色素 P-450 形成乙烯。
DOI：
--
发表时间：
1988
期刊：
Drug metabolism and disposition: the biological fate of chemicals
影响因子：
--
作者：
Ding,XX;Coon,MJ
通讯作者：
Coon,MJ
Sasaki, M.：“大肠杆菌recC 基因的核苷酸序列分析”J. Biochem。
DOI：
--
发表时间：
--
期刊：
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1073/pnas.82.12.4065
发表时间：
1985-01-01
影响因子：
11.1
作者：
KOOP, DR;CRUMP, BL;COON, MJ
通讯作者：
COON, MJ