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.
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.
Ohgiya
S
S
Hokkaido National Industrial Research Institute, Agency of Industrial Science and Technology, Toyohira-ku, Sapporo, Japan. ohgiya@hniri.go.jp
Goda
T
T
Hoshino
T
T
Kamataki
T
T
Ishizaki
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017931:DNA Primers; D016681:Genome, Fungal; D006168:Guinea Pigs; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008322:Mammals; D008647:Mesocricetus; D051379:Mice; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1; D009251:NADPH-Ferrihemoprotein Reductase