Cynomolgus Monkey Cytochrome P450 2C43: cDNA Cloning, Heterologous Expression, Purification and Characterization

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摘要：

The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b5 and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4′-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4′-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.

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关键词：

cynomolgus monkey CYP2C43 heterologous expression liver microsomes reconstitution DLPC, dilauroylphosphatidylcholine APMSF, 4-(2-aminoethyl)benzenesulfonyl fluoride HCl CYP, cytochrome P450 IPTG, isopropyl-β-d-thiogalactopyranoside