Development Of Human CYP3A4 Two Non-synonymous Mutations Yeast Expression System And Characterization Of The Polymorphism Effecting On Metabolic Analysis And Drug Screen In Vitro

Human Cytochrome P450 (CYP) is a superfamily of heme-containing mixed-function oxygenases, which exist primarily in the lipid bio-layer of the endoplasmic reticulum of hepatocytes. It participate in metabolizing a vast array of endogenous substrate like fatty acids, steroids and vitamins as well as exogenous substrate like drugs, dietary substance and environmental pollutants. Accroding to the difference of nucleotide sequence homology, CYPs can be divided into many subfamily and CYP3A account for 30%. Compare to other subfamily, not only the expression of apoenzyme is more, but the forms of the presence are varied. CYP3A4 is the most abundant isoform of CYP in adult human liver and intestinal tract, CYP3A4 is responsible for the metabolism of about 50% of commonly clinical drugs through oxidation, peroxidation, and reduction. The variable expression of CYP3A4 is at least partially due to the factors below, including induction by drugs, endogenous compounds, and environmental chemicals, but also includes genetic factors. Several studies suggested that 30～85% of the interindividual variability in CYP3A is predominantly due to genetic factors, such as non-synonymous single nucleotide polymorphisms (SNPs).We have already known many SNPs of CYP3A4.Some of them contribute to the alteration of CYP3A4 enzyme activity. Two novel mutations of CYP3A4 were published in http://www.cypalleles.ki.se, CYP3A4 (F176V) and CYP3A4 (I223R). But no one has researched on the affection of the two new SNPs. We introduced the mutations into the c DNA of the wild type by site-specific PCR. Then the two CYP3A4 genes were sub-cloned into a yeast expression vector pYES2/CT and were chosen by the ampicilin. Following sequencing, we transported the recombinants to the yeast strain which contained NADPH-cytochrome P-450 reductase (CPR). After that we used galactose to induce the P450 enzymes expressed in yeast.Fractions that contained recombinant 3A4 enzyme were isolated by differential centrifugation. The Km, Vmax and CLint of the enzyme were determined by the unique fluorescent substrate, dibenzylfluorescein (DBF)-O-dealkylation.From the results we could concluded, DBF could be metabolized by the two allelic enzymes. Compared to the wildtype, Km of the mutation are increase, Vmax of the mutation are decrease, CLint of the mutation are also decreas. Our study lay the foundation for the further research.