Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae with the copper-inducible CUP1 promoter.
The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) in Saccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and the ADH1 promoter, 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 the CUP1 promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed in 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 from N-nitrosodiethylamine and for aniline p-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions. Furthermore, 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.
Fujita
V S
VS
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
Thiele
D J
DJ
Coon
M J
MJ
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003300:Copper / Q000494:pharmacology; D019327:Copper Sulfate; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004790:Enzyme Induction / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D008668:Metallothionein / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D003300:Copper; D003577:Cytochrome P-450 Enzyme System; D008668:Metallothionein; D006899:Mixed Function Oxygenases; D000426:Alcohol Dehydrogenase; D019392:Cytochrome P-450 CYP2E1; D019327:Copper Sulfate