The Establishment Of Human Cyp1a2 And Cyp2e1 Wild-type Yeast Expression System And The Cyp1a2 Fluorescence Drug Screening Platform

The human cytochrome P450(CYP450) superfamily members metabolize a vast variety of clinically,physiologically,and toxicologically importante compounds.CYP1 A2 is the one of most important isoform of CYP in human liver.It is about 15%of total human liver CYPs,the enzyme content of CYP1A2 ranks third.CYP1A2 involved in metabolism of many precarcinogen to activate the role,CYP1A2 participates wide range of drug metabolism,as well as responsible for the hydroxylation reaction of a number endogenous hormone.There is great difference of CYP1A2 activity between individuals, CYP1A2 genetic polymorphism may affect individual susceptibility to cancer and may cause some drugs the individual differences in treatment effect.CYP2E1 is also an important precarcinogen- metabolizing enzyme,the main substrates are lipophilic small molecule compounds,and CYP2E1 can be induced by ethanol and other small molecules.To develop a broadly applicable assay system for studying human CYP1A2,we cloned the cDNA of CYP1A2 into pYES2/CT vector for galactose-inducible expression in budding yeast Saccharomyces cerevisiaee,which was already integreted with CYP450 Oxidoreductase (POR) gene.Transformed yeasts produced large quantities of microsome-bound1A2 enzymes as determined by Western analysis;a 55 kDa protein was detected.The isolated S9 microsomes were used to measure the kinetic constants of 1A2 enzymes in real-time assays using a fluorogenic substrate CEC.It showed that the recombinant 1A2^*1A enzyme possess evident activity,the data can be used for non-linear regression analysis method to calculate the value of Km and Vmax.we tested the inhibition of the recombinant CYP1A2^*1A by a known inhibitor 7,8-benzoflavon in the Fluorescence Assays;then we chose the specific inhibitors of other CYPs(2C8/2C9/2C19/2D6/3A4) to do the competitive trials,the results indicated the IC₅₀ of 7,8-benzoflavon is far lower than other inhibitors except Tranylcypromine.Analysis of this study confirmed the establishment of the CYP1A2 fluorescent drug screening platform is feasible.7 kinds of antidepressant drugs used for drug screening fluorescence experiments to study the inhibition of CYP1A2 obtaind from our yeast expression system.This study provides experimental data in vitro for the clinical use of antidepressant drugs in combination. In addition,the study also established the CYP2E1 wild-type yeast expression system, confirmed through sequencing that the CYP2E1^*1A consensus cDNA sequence,identified the protein expression by Western blot technology,to lay the foundation for the establishment of drug screening platform.