Expression of human CYP2E1 in insect cells using bac-to-bac expression system.
- Author:
Ke LU
1
;
Su ZENG
;
Tong-wei YAO
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Baculoviridae; genetics; metabolism; Cytochrome P-450 CYP2E1; biosynthesis; genetics; Escherichia coli; genetics; Genetic Vectors; genetics; Humans; Recombinant Fusion Proteins; biosynthesis; genetics; Spodoptera; genetics; metabolism; Transfection
- From: Journal of Zhejiang University. Medical sciences 2008;37(2):118-125
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo obtain recombinant human CYP2E1 and to determine its activity by using the specific probe substrate.
METHODSCYP2E1 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.
RESULTThe 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.
CONCLUSIONActive recombinant CYP2E1 has been obtained by bac-to-bac expression system and its activity is similar to previous reports.
