Expression And Characterization Of Human Recombinant CYP1A2 And CYP3A4

Most drugs are the exogenous compounds, most of which need some structural modification to be excreted out of the body. This modification process is called "metabolism". Cytochrome P450 (CYP) is a superfamily of monooxygenases that play an important role in the metabolism of various endogenous and exogenous compounds. More than 90% of the drugs are metabolized by the hepatic microsome CYPs, which have the characteristics of wide substrates profile, low specificity, big structure differences, big individual variance, and being easy to be induced and inhibited. As a result, either P450 inhibition or induction in patients receiving multiple medications has the potential to precipitate clinically important drug interactions. Whereas P450 inhibition may lead to adverse effects as aresult of substantial increases in systemic drug concentrations, P450 induction could compromise the pharmacological efficacy of therapeutic agents due to significant decreases in their circulating concentrations. The FDA has released the governmental documents for studis of drug metabolism and drug interation to decrease the risk of drug development and make the safety evaluation of drug candidates in the early stage.In recent years, animal systems have been used widely in drug metabolismstudies because of the limitation of using human stuff from the ethics point. As we all know, there are differences between animals and human beings. So the data from the animal systems can not directly indicate the characteristics of human P450s. Futher more, it is difficult to obtain the purified human P450 isoforms. Fortunately, cloning and expression single drug metabolizing enzyme in vitro has been widely used, and most of CYPs have been expressed in vitro. For example, many CYPs have also been expressed with E.coli, yeast, baculovirus-insect cells and mammalian cells. Nowadays, expressing heterologous proteins with baculovirus in insect cells are widely used, due to its combination of large yield of protein and the high activity.This project is utilizing the baculo virus-insect cells system to express two vital members of CYP isoforms CYP1A2 and CYP3A4, then to investigate the metabolism activity with the recombinant protein to drugs or compounds. A single CYP isoform will be obtained if the gene components have been identified. On this condition, it is possible for us to research the phase I metabolism function of some CYP isoform, learn about the oxidation of the compounds, and predict the degree of the phase I metabolism and interaction of the compounds. 1 Cloning, expression and characterization of human CYP1A21.1 Cloning of the gene CYP1A2 and construction of the recombinant virusesThe cDNA of the CYP1A2 was transcripted from RNA of a Chinese human liver by RT-PCR. EcoRI site was introduced at the 5' terminus whereas a His-tag andXhoI sites were added to 3' terminus. The PCR products were T-A cloned into the pGEM-T vector and then were screened by restriction enzyme digestion and DNA sequencing. The results confirmed this instered gene had the desired CYP1A2 gene sequence. The recombinant pGEM-CYPlA2 was amplified by tansforming into E.coli DH5a.The target gene segment by EcoRI and Xhol digestion was inserted into the vector pFastBac which is also cleaved with these two endonucleases. The harvested recombinant pFastBac-CTPi^ was then transformed into E.coli DHlOBac in which recombinant Bacmid (Bacmid-CTPL42) would generate by transposition. After Bacmid-CYP 1A2 transfected the Sf9 cell, the recombinant virus would generate.1.2 Co-expression of the recombinant CYPlA2-ORThe recombinant viruses were amplified with MOI 0.05 ~ 0.1 until the viral titer reached 108pfu/mL or higher. The recombinant viruses of CYP1A2 was mixed with the ones of CYPOR in volumn ratio of 9:1 and then infect the Sf9 cells with MOI 20 to express the recombinant CYP1A2-OR. The infected Sf9 cells were harvested after 96h. The collected cells were socinated and the suspension of recombinant CYP1A2-OR protein was obtained through standard ecntrifugation. The activity would be identified after the protein concentration was assayed by the method of BCA.1.3 Validation of the recombinant CYP1A2 transcriptionThe total RNA was isolated from infected Sf9 cells and the transcriptiono f recombinant CYP1A2 mRNA was identified by RT-PCR, with the parent Sf9 cells as control. As a result, the CYP1A2 was transcripted in the infected Sf9 cells, but not in parent Sf9 cells.1.4 Western blotting analysis of recombinant CYP1A2The His-tag in the protein allowed it to be detected by Western blotting. The result displayed that recombinant CYP1A2 protein can be recognized by mouse anti-His monoclonal antibodies. It approved CYP1A2 gene had been inserted into the expression vector correctly and expressed successfully.1.5 Activity assay of the recombinant CYP1A2-ORThe activity assay for recombinant CYP1A2-OR was conducted by HPLC with phenacetin as its substrate. The result indicated that the recombinant CYP1A2-OR can catalyze the phenacetin to be O-deethylated significantly and but not the parent Sf9 cells. The values of apparent Km, Fmax and Clint of the recombinant CYP1A2-OR to phenacetin were 82.04±11.39 μmol/L (n=3), 1.78±0.26 nmol-min^-mg"1 protein (n=3) and 0.02 mL-mnV'-mg^protien respectively assayed in a series of substrate concentrations.2 Cloning, expression and characterization of human CYP3A4 2.1 Cloning of the gene CYP3A4 and construction of the recombinant virusesThe cDNA of the CYP3A4 was transcripted from RNA of a Chinese human liver by RT-PCR. NotI site was introduced at the 5' terminus whereas a His-tag and Xholsites were added to 3' terminus. The PCR products were T-A cloned into the pGEM-T vector and then were screened by restriction enzyme digestion and DNA sequencing. The results confirmed this instered gene had the desired CYP3A4 gene sequence. The recombinant pGEM-CYP3A4 was amplified by tansforming into E.coli DH5a.The target gene segment by NotI and Xhol digestion was inserted into the vector pFastBac which is also cleaved with this two endonucleases. The harvested recombinant pFastBac-CTPi^ was then transformed into E.coli DHlOBac in which recombinant Bacmid (Bacmid-CTPJ^/) would generate by transposition. After Bacmid-CyPi^ transfected the Sf9 cell, the recombinant virus would generate.2.2 Co-expression of the recombinant CYP3A4-ORThe recombinant viruses were amplified with MOI 0.05~0.1 until the viral titer reached 108pfu/mL or higher. The recombinant viruses of CYP3A4 was mixed with the ones of CYPOR in volumn ratio of 1:1 and then infect the Sf9 cells with MOI 20 to express the recombinant CYP3A4-OR. The infected Sf9 cells were harvested after 96h. The collected cells were socinated and the suspension of recombinant CYP3A4-OR protein was obtained through standard ecntrifugation. The activity would be identified after the protein concentration was assayed by the method of BCA.2.3 Validation of the recombinant CYP3A4 transcriptionThe total RNA was isolated from infected Sf9 cells and the transcriptiono f recombinant CYP3A4 mRNA was identified by RT-PCR, with the parent Sf9 cells as control. As a result, the CYP3A4 was transcripted in the infected Sf9 cells, but not in parent Sf9 cells.2.4 Western blotting analysis of recombinant CYP3A4The His-tag in the protein allowed it to be detected by Western blotting. The result displayed that recombinant CYP3A4 protein can be recognized by mouse anti-His monoclonal antibodies. It approved CYP3A4 gene had been inserted into the expression vector correctly and expressed successfully.2.5 Activity assay of the recombinant CYP3A4-ORThe activity assay for recombinant CYP3A4-OR was conducted by HPLC with9testosterone as its substrate. The result indicated that the recombinant CYP3A4-OR can catalyze the testosterone to be hydroxylated significantly and but not the parent Sf9 cells. The values of apparent .Km, Fmax and Clint of the recombinant CYP3A4-OR to testosterone were 45.86±10.88 umol/L (n=3), 0.10±0.03 nmol-min^-mg'1 protein (n=3) and 0.002 mL-min"1-mg'1 protein respectively assayed in a series of substrate concentrations.The main metabolite of testosterone via recombinant CYP3A4-OR was identified by HPLC-MS and the result confirmed that it is a hydroxylated product of testosterone. In addition, the inhibition assay was conducted with ketoconazole as specific inhibitor. As a result, more than 70% of the recombinant CYP3A4-OR catalytic activity for testosterone was inhibited significantly with ^mol/L ketoconazole.As a conclusion, we successfully expressed the recombinant CYP1A2 and CYP3A4 with catalytic activity by the baculovirus expression system—Bac-to-Bac system. And the values of Km and Fniax of the recombinant CYP1A2 toward phenacetin were 82.04±11.39 umol/Land 1.78±0.26 nmol-min^-mg"1 protein, whereas the values of Km and Fhiax of the recombinant CYP3A4 toward testosterone were 45.86±10.88 μmol/L and 0.10±0.03 nmol-min^mg"1 protein. The enzymes CYP1A2 and CYP3A4 showed the good catalytic activity, and the results in our experiment were in accordance with them reported on the whole. It approved that these two recombinant CYPs can be utilized in the further in vitro study of drug metabolism.