Ce. Ong et al., BACULOVIRUS-MEDIATED EXPRESSION OF CYTOCHROME P4502C8 AND HUMAN NADPH-CYTOCHROME P450 REDUCTASE - OPTIMIZATION OF PROTEIN EXPRESSION, Xenobiotica, 28(2), 1998, pp. 137-152
1. High expression levels of cytochrome P450 (CYP) 2C8 and NADPH-cytoc
hrome P450 oxidoreductase (OxR) in Spodoptera frugiperda (Sf21) cells
have been achieved using the baculovirus expression system. 2. The bac
ulovirus dual expression plasmid, pAcUW31, was used to insert CYP2C8 a
nd OxR cDNAs downstream of the polyhedrin (polh) or p10 promoters, eit
her separately or together, generating four recombinant baculoviruses;
two expressing single proteins (CYP2C8 driven by the p10 promoter, bV
p10.2C8 or OxR driven by the polh promoter, bVpolh.OxR) with another t
wo coexpressing both CYP2C8 and OxR under reciprocal control of the po
lh and p10 promoters (bVpolh.OxR-p10.2C8 and bVpolh.2C8-p10.OxR). 3. H
igh levels of singly expressed CYP2C8 and OxR were achieved from bVp10
.2C8 and bVpolh.OxR, with levels of 0.7-1.2 nmol CYP/mg protein and 40
0-500 nmol cytochrome c reduced/min/mg protein respectively. 4. The tw
o dual gene clones (bVpolh.OxR-p10.2C8 and bVpolh.2C8-p10.OxR) showed,
in general, greater variation in CYP content and OxR activity than si
ngle gene clones. Screening was therefore necessary for the selection
of dual gene clones expressing both proteins optimally. 5. Sf21 micros
omes infected by selected dual gene clones were, on average, 14 times
more active in tolbutamide hydroxylase activity than those expressing
CYP2C8 alone, with a mean spectral CYP content of 79 pmol/mg cell lysa
te protein and a mean OxR level of 600 nmol/min/mg cell lysate protein
.