ISSN: 0973-7510

E-ISSN: 2581-690X

He Huang1, Juanli Zhu1,2, Huijuan Wang and Chao Chen
1College of Life Science, Northwest University, Xi’an – 710 069, P. R. China.
2National Engineering Research Center for Miniaturized Detection Systems, Northwest University, Xi’an – 710 069, P. R. China.
J Pure Appl Microbiol. 2013;7(2):1331-1338
© The Author(s). 2013
Received: 30/03/2013 | Accepted: 22/05/2013 | Published: 30/06/2013
Abstract

To develop a broadly applicable assay system for studying human CYP1A2, we cloned the cDNA of CYP1A2*1A (wild-type) into pYES2/CT vector for galactose-inducible expression in budding yeast Saccharomyces cerevisiae, which was already integrated with CYP450 Oxidoreductase (POR) gene. Transformed yeast produced large quantities of microsome-bound CYP1A2*1A enzymes as determined by western blotting analysis; a 55 kDa protein was detected. The isolated S9 microsomes were collected to measure the kinetic constants of CYP1A2*1A enzymes in real-time assays using a fluormetric substrate CEC. It showed that the recombinant CYP1A2*1A enzyme possess evident activity, the Km value was about 7ìmol/L; we tested the inhibition of the recombinant CYP1A2*1A by a known inhibitor 7, 8-benzoflavon in the fluorescence assays; then we chose the specific inhibitors of other CYPs (2D6/3A4/2C8/2C9/2C19) to do the comparative trials, the results indicated the IC50 of 7, 8-benzoflavon is far lower than other inhibitors. Our study validated the feasibility of the established in vitro CYP1A2 detection system, which can guide the further study of high-throughput drug screening and drug-drug interaction.

Keywords

CYP1A2*1A (wild-type), Enzyme Kinetics, Fluorescence Assay, Inhibition Assay, 3-Cyano-7-ethoxycoumarin (CEC), 7, 8-benzoflavon

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