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.
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.
Rao
K N Purnachandra
KN
Premas Biotech Pvt. Ltd., IMT Manesar, Gurgaon, Haryana, India.
Suman
Sunil Kumar
SK
Kiran
Y Ravi
YR
Kuanr
Arup Ranjan
AR
Gupta
Anuj Kumar
AK
Bhalla
Kunal
K
Kumar
Vipul
V
Kundu
Prabuddha
P
Arora
Kajal
K
Soni
Rajeev
R
D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000172:deficiency* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013426:Sulfaphenazole / Q000494:pharmacology
D011994:Recombinant Proteins; D013426:Sulfaphenazole; D015786:Cytochromes b5; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase; D010447:Peptide Hydrolases; D019793:Fluorescein