1. Co-expression of recombinant human CYP2C9 with human cytochrome P450 reductase in protease deficient S. cerevisiae strain at a higher scale yields an enzyme of higher specific activity.
- Author
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Rao KN, Suman SK, Kiran YR, Kuanr AR, Gupta AK, Bhalla K, Kumar V, Kundu P, Arora K, and Soni R
- Subjects
- Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases biosynthesis, Aryl Hydrocarbon Hydroxylases genetics, Cytochrome P-450 CYP2C9, Cytochromes b5 genetics, Cytochromes b5 metabolism, Fluorescein metabolism, Humans, Kinetics, Microsomes enzymology, NADPH-Ferrihemoprotein Reductase biosynthesis, NADPH-Ferrihemoprotein Reductase genetics, Peptide Hydrolases genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Substrate Specificity, Sulfaphenazole pharmacology, Aryl Hydrocarbon Hydroxylases metabolism, NADPH-Ferrihemoprotein Reductase metabolism, Peptide Hydrolases deficiency, Saccharomyces cerevisiae enzymology
- Abstract
Cytochrome P450 (CYP450) isozymes play an important role in the study of drug metabolism and drug discovery. A number of reports are available that describe recombinant expression of CYP450 isozymes. In this paper, human CYP2C9 and human cytochrome P450 reductase cDNAs were cloned and expressed in Premas proprietary yeast episomal and integrative vectors respectively under the influence of GAL1 promoter. Yeast cells were grown and induced at optimal parameters to make microsomal membranes. Isolated microsomal membranes were analyzed for CYP2C9 and cytochrome P450 reductase activity, CYP2C9 content and inhibition properties. We report heterologous expression of human CYP2C9 along with human cytochrome P450 reductase in protease deficient S. cerevisiae at a 5 litre scale resulting in high yields (8-10 nmols/litre) of enzyme with higher specific activity (2-3 fold higher). This yields a superior enzyme and makes it amenable to miniaturization of screening assays with concomitant lowering of costs.
- Published
- 2010
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