| pubmed-article:9434754 | pubmed:abstractText | Expression of recombinant cytochrome P450s (P450s) in mammalian cells has been used as a powerful tool to study these enzymes. However, the activity of CYP3A4 expressed in several stable mammalian cell lines was much lower than native enzyme in human liver. The low level of recombinant CYP3A4 may have been due to the low copy number of the cDNA. In addition, the low activity is caused by the low level of P450 reductase in these cells. To achieve high levels of CYP3A4 expression, we employed gene amplification of the CYP3A4 cDNA in Chinese hamster ovary (CHO) cells followed by transfection of the P450 reductase cDNA. Using this strategy, we have obtained a cell line, designated D3A4, with high levels of recombinant CYP3A4. The content of spectrally active P450 was 14 pmol/mg total cellular protein. Hemin treatment increased the P450 content 2-fold. Upon coexpression of P450 reductase in DHR/3A4 cells, enzyme activity of CYP3A4 was stimulated 15-fold, despite a 40% decrease in spectrally active P450. Interestingly, the latter effect was not due to a decrease in CYP3A4 mRNA. Treatment of these cells with hemin, however, counteracted the P450 reductase-mediated decrease of spectrally active P450. These data demonstrate that P450 reductase has a strong influence on the levels of recombinant P450 holoenzyme, possibly by modulating the level of heme in CHO cells. Concomitantly our results show that the gene amplification strategy provides a powerful approach to obtain a high level of functional recombinant P450. | lld:pubmed |
