Cloning and expression of heterologous human cytochrome P450 3A4-NADPH P450 reductase in Escherichia Coli / Ainul Zahiah Abdul Malik

Cytochrome P450 3A4 (CYP3A4) is one of the Cytochrome P450 monooxygenases, dominant in drug metabolism in human liver by virtue of its high levels of expression in liver and other tissues. It represents about 30% of total CYP450. The aim of this study is to develop a heterologous in vitro drug metabolizing enzyme system for human CYP3A4 with NADPH-P450 reductase using Escherichia coli (E. coli). The advantages of this bacteria system include low maintenance cost, ease of use and the high yield of enzymes. The gene encoding CYP3A4 was identified from database and specific primers were designed. The cDNA was then amplified using polymerase chain reaction and the human liver cDNA was the initial template. The recombinant 17αCYP3A4 with the NADPH-P450 reductase expression plasmid was constructed for cloning into E. coli. The two plasmids were co-expressed and harvested. The identification and confirmation of the protein was done by SDS-PAGE, Western-Blot and spectral analysis; while the functionality of the enzyme was performed using Vivid P450 kit. The CYP3A4 gene sequence and its construct in the expression plasmid were confirmed with direct sequencing. The optimal condition for large scale expression of CYP3A4 protein was 30 ˚C for 48 hours. The pro tein expressed was successfully characterized. The SDS-PAGE and Western-blot confirmed the bands at 57 kDa for CYP3A4 and 80 kDa for NADPH-P450 reductase. The spectral determination of CYP3A4 showed a absorbance peak at the UV wavelength of 450 nm. The yield of this recombinant protein was 71.4286 nmol/ml protein; and the activity was 9442.89 nmol/min/ml protein. The Michaelis-Menten parameters determined from the protein were 35.2 µM for the Km and 1366.3 RFU/min/pmol CYP3A4 for the Vmax. An in vitro heterologous system containing co-expression of CYP3A4 with NADPH-P450 reductase in E. coli was successfully developed and characterized. The system would be of enormous benefit for elucidation of metabolic pathways for new chemical entities as well as in the study of drug-drug and drugherb interactions.