Expression of a human P-450IIC gene in yeast cells using galactose- inducible expression system

T Yasumori, N Murayama, Y Yamazoe, A Abe, Y Nogi, T Fukasawa and R Kato

Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.

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.

Volume 35, Issue 4, pp. 443-449, 04/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

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