Expression of cytochrome P450 2C9 (CYP2C9) in escherichia coli and its functional characterization

Boon Hooi Tan, Yan Pan, Uma Devi Palanisamy, Iekhsan Othman, Nafees Ahmed, Beow Chin Yiap, Chin Eng Ong

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    This study aimed to express the major human hepatic drug metabolizing cytochrome P450 (CYP), CYP2C9, together with NADPH cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate its catalytic activities. Co-expression of CYP2C9 and OxR was achieved by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates valsartan and tolbutamide. Results from immunoblotting demonstrated the presence of CYP2C9 protein in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to previously published data. Kinetic parameters, Km and Vmax values determined from the valsartan and tolbutamide hydroxylase assays, were also concordant with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP2C9 suitable for drug metabolism and interaction studies.

    Original languageEnglish
    Pages (from-to)43-55
    Number of pages13
    JournalAsia-Pacific Journal of Molecular Biology and Biotechnology
    Volume27
    Issue number1
    DOIs
    Publication statusPublished - 2019

    Keywords

    • Cytochrome P450
    • Escherichia coli
    • Heterologous expression
    • Human CYP2C9
    • Kinetic analysis

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