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  • Title: Establishment of a novel host, high-red yeast that stably expresses hamster NADPH-cytochrome P450 oxidoreductase: usefulness for examination of the function of mammalian cytochrome P450.
    Author: Ohgiya S, Goda T, Hoshino T, Kamataki T, Ishizaki K.
    Journal: Arch Biochem Biophys; 1997 Jul 15; 343(2):215-24. PubMed ID: 9224733.
    Abstract:
    A novel strain of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal cytochrome P450s was established and named High-red yeast. Hamster NADPH-cytochrome P450 oxidoreductase (P450 reductase) cDNA to be introduced into yeast was isolated from a hamster liver cDNA library. The cDNA was 2421 bp long and contained an entire coding region for 667 amino acids. The NH2-terminal amino acid sequence deduced from the hamster P450 reductase cDNA was identical with that of the enzyme purified from hamster livers except for deletion of the initial methionine. A delta-sequence derived from yeast retrotransposon Ty was cloned and used as a sequence for homologous recombination in a yeast genome. S. cerevisiae YPH500 was transformed with a multi-integration cassette containing the expression unit of the hamster P450 reductase and the delta-sequence. The transformant showing the highest activity of the P450 reductase was named High-red yeast. High-red yeast carried more than six copies of the multi-integration cassettes in a single chromosome and retained the multi-integration cassettes over a period of 100 generations under nonselective culture conditions, indicating that this yeast was a mitotically stable transformant. The microsomes prepared from High-red yeast had 20 times the P450 reductase activity of the microsomes prepared from the parental yeast. Due to the high activity of the hamster P450 reductase, the 7-ethoxycoumarin deethylase activity of mouse CYP1A1 expressed in High-red yeast was 250 times higher than the activity of mouse CYP1A1 expressed in the parental yeast.
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