Expression And Activity Assessment Of CYP2C9 Ezyme

Cytocrome P₄₅₀ enzymes are mono-oxigenases wildely existed andplayed a key role in phase 1 of drug motablism. CYP2C9 is a kind of P₄₅₀responsble for important lipofilic drugs such as warfarin, tobultomide andphenytoin. Reconbinant Human CYP2C9 are of great sense very important instructural and functional research and determination of main enzymes ofclearance of new drugs. Our research is to express human Cyp2C9 in COS-7cells. Plasmids PREP9-CYP2C9 are digested by Kpn1 and Xho1 and CYP2C9gene fragments are extracted to inserted in another plasmid pcDNA3.1+ usingT4 ligase. Reconbinat pcDNA3.1+-CYP2C9 was amplified in E.coli Top10.Endo-toxity free plasmid were extracted to trasfect COS-7 cells by liposome tomake a transient expression. RT-PCR results showed that CYP2C9 gene hadbeen transcriped in COS-7 cells. Immunoblot analysis proved that CYP2C9proteins existed in recombinant cells. To meassure the activity of CYP2C9enzymes, tolbutamide was chosen as a prob drug and 4-OH-tobutamide wastested by HPLC and Mass Spectrum. The recombinant CYP2C9 did haveoxigenases activity but not very high. In our further research, NADPH-P-450reductase will be added in the reaction system to increase the activity ofCYP2C9. What's more, the reaction will be quantified to determine the Kmand Vmax value of reconbinant CYP2C9 enzymes.