Expression of a human P-450IIC gene in yeast cells using galactose-inducible expression system.
A cDNA of a human liver cytochrome P-450, corresponding to P-450 human-2, was expressed in Saccharomyces cerevisiae cells by the use of a galactose-inducible expression vector containing the GAL7 promoter and terminator. In Western blots using anti-P-450 human-2 IgG, a single band, which exhibited mobility identical to that of authentic P-450 human-2 purified from human liver, was detected in microsomes of the yeast cells. The amount synthesized in yeast was estimated to be approximately 1% of the total cell protein, and approximately 25% of the cytochrome existed in the holoenzyme state. Microsomes from the P-450 human-2-producing yeast showed a catalytic activity towards benzo(a)pyrene, and the activity was significantly enhanced by the addition of purified NADPH-cytochrome P-450 reductase. The yeast microsomes also catalyzed (S)-mephenytoin 4-hydroxylation but not the demethylation. The present results indicate that the yeast cells containing P-450 human-2 cDNA synthesize a functionally active form of the enzyme, the chemical and catalytic properties of which are identical to those of the human liver preparation.
Yasumori
T
T
Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
Murayama
N
N
Yamazoe
Y
Y
Abe
A
A
Nogi
Y
Y
Fukasawa
T
T
Kato
R
R
D000937:Antigen-Antibody Reactions; D001189:Aryl Hydrocarbon Hydroxylases; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D005690:Galactose / Q000494:pharmacology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D006827:Hydantoins / Q000378:metabolism*; D008617:Mephenytoin / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D006827:Hydantoins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D008617:Mephenytoin; D005690:Galactose