Drug Metabolism Letters

Zhiyang Zhao
Amgen
Cambridge, MA
USA

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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(s): K. N. Purnachandra Rao, Sunil Kumar Suman, Y. Ravi Kiran, Arup Ranjan Kuanr, Anuj Kumar Gupta, Kunal Bhalla, Vipul Kumar, Prabuddha Kundu, Kajal Arora, Rajeev Soni.

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 (8-10nmols/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.

Keywords: CYP2C9, heterologous expression, high yield, inhibition studies, microsomal membranes, protease deficient, S. cerevisiae, Recombinant Human CYP2C9, isozymes, drug metabolism, drug discov-ery, reductase activity, miniaturization, endoplasmic reticulum, redox regulatory partner, drug-drug interac-tions, inflammatory drugs, post-translational modifications, multi-subunit proteins, host-specific proteases, high-throughput manner, Restriction endonucleases, cose-6-phosphate dehydrogenase, tryptone, leucine, adenine, lysine, uracil, dithiothreitol, bovine serum albumin (BSA), glass homogenizer, Enzyme Kinetics

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Article Details

VOLUME: 4
ISSUE: 4
Year: 2010
Page: [246 - 253]
Pages: 8
DOI: 10.2174/187231210792928233
Price: $58