Expression Of Human Recombinant CYP3A4 And CYP2J2 And Its Related Metabolism Research

Cytochrome P450(CYP450)enzymes are a protein superfamilies containing heme and metabolize about 75%of clinical drugs.Recombinant CYP450 plays an irreplaceable role in the study of CYP450-mediated metabolism as an important in vitro model for drug development and safety assessment.However,the expression system of recombinant CYP450 has certain defects,such as simple mechanism of protein modification,high cost and long culture period.We established a highly efficient and stable method for the expression of human recombinant CYP450 by the Drosophila expression system.In this thesis,we constructed human recombinant CYP3 A4 and 2J2.First,stable cell lines were obtained by constructing recombinant plasmids,stable transfection and screening by blasticidin.We established CYP3A4,CYP2J2,OR and CYB5A cell lines,OR/CYB5A/CYP3A4(or CYP2J2)co-expressing cell lines.Then,the factors influencing the protein expression were optimized,containing the inducer and its concentration and the concentration of heme.The stable cell lines were cultured in large scale and prepared into recombinant microsomes,which activities were detected by liquid chromatography tandem mass spectrometry.We obtained human recombinant CYP3A4(Km,71.0 ?M;Vmax,0.4962 nmol/min/mg protein)and CYP2J2(Km,1.65 ?M;Vmax,0.6630 nmol/min/mg protein)microsomes.The metabolism of the above microsomes was studied using the positive inhibitors.We further screened the inhibitory effect of 50 natural compounds against CYP2J2.We found that plumbagin was a mixed-type inhibitor against CYP2J2(IC50,1.17 ?M;Ki,1.06 ?M).In conclusion,this thesis established a simple and efficient method for expression of human CYP3A4 and CYP2J2 enzymes.In addition,we found that plumbagin was an inhibitor of CYP2J2.It had a certain role on promoting metabolism research of CYP2J2.