[Expression of human CYP2E1 in insect cells using bac-to-bac expression system].
To obtain recombinant human CYP2E1 and to determine its activity by using the specific probe substrate.



CYP2E1 cDNA was obtained by RT-PCR using human liver RNA as template. The cloned CYP2E1 cDNA was ligated with pFastBac vector to generate recombinant pFastBac-CYP2E1, which was then transformed into E. coli DH 10 Bac. Recombinant Bacmid-CYP2E1 was generated by transposition. Then Spodoptera frugiperda (Sf9) insect cells was infected with Bacmid-CYP2E1 to generate recombinant baculoviruses carrying human CYP2E1 cDNA. Finally, Sf9 insect cells were triinfected with recombinant baculoviruses carrying human CYP2E1, CYPOR and CYPb5. The activity of the recombinant enzymes was determined using chlorzoxazone as the substrate.



The Kmand Vmaxof recombinant CYP2E1 to chlorzoxazone was (72.4 +/-8.7) micromol. L(-1) and (2.41 +/-0.10) micromol.min(-1)?g(-1)protein, respectively.



Active recombinant CYP2E1 has been obtained by bac-to-bac expression system and its activity is similar to previous reports.
Lu
Ke
K
Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, China.
Zeng
Su
S
Yao
Tong-wei
TW
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D019392:Cytochrome P-450 CYP2E1