Heterogenous expression and activity analysis of human cytochrome P450 3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18
10.3867/j.issn.1000-3002.2009.06.007
- VernacularTitle:人细胞色素P450 3A4突变体CYP3A4.3, CYP3A4.4, CYP3A4.5和CYP3A4.18重组酶的异源表达与活性
- Author:
Xiaowen WANG
;
Su ZENG
- Publication Type:Journal Article
- Keywords:
cytochrome P- 450 CYP3A4;
mutation;
Baculoviridae;
testosterone
- From:
Chinese Journal of Pharmacology and Toxicology
2009;23(6):456-463
- CountryChina
- Language:Chinese
-
Abstract:
AIM To express recombinant human cytochrome P450 3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18, and to employ them for in vitro metabolism studies of CYP3A4. METHODS Use Bac-to-Bac baculovirus expression system to recombinant baculovirus carrying cDNA of CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18. Spodoptera frugiperda 9 (Sf9), cells were co-infected by recombinant viruses of CYP3A4 mutants, human NADPH-P450 oxidoreductase and cytochrome b5 to obtain recombinant proteins CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity. RESULTS The mRNA transcription of CYP3A4 mutants in Sf9 cells were validated by RT-PCR. Testosterone and 7-benzyloxy-4-(trifluoromethyl) coumarin were metabolized by the lysates of Sf9 cells infected by the recombinant viruses. CONCLUSION CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity were successfully expressed by baculovirus-insect cell expression system. The results indicated that recombinant CYP3A4. 5 showed lower activity comparing to the wild type protein towards testosterone, while CYP3A4. 18 with higher activity, and for CYP3A4.3 and CYP3A4.4 showing similar activity to the wild type protein.
