Baculovirus-directed expression of rabbit UDP-glucuronosyltransferases in Spodoptera frugiperda cells.
The rabbit liver UDP-glucuronosyltransferase (UGT) cDNAs that encode the 4-hydroxybiphenyl UGT2B13 and 4-nitrophenol UGT1A6 have been cloned into baculovirus. Spodoptera frugiperda (SF-9) cells infected with the UGT recombinant baculovirus produced significant amounts of protein, which was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and pulse chase with 35S-amino acids. The expression of the UGT proteins in SF-9 cells were detected at approximately 24 hr postinfection, with maximal levels of protein seen at 48 hr. Immunoprecipitation of newly synthesized 35S-labeled proteins demonstrated that the maximal rate of protein synthesis in SF-9 cells infected with the UGT baculovirus occur at 48 hr postinfection, although the proteins are abundant in the cells for up to 96 hr. When compared with the expression levels of the same cDNAs through transient transfection into COS-1 cells, the insect-derived UGT proteins showed nearly 50- to 100-fold greater protein accumulation. Although kinetic analysis demonstrated that turnover rate of the SF-9-expressed proteins were greater than their counterparts in COS-1 cells, K(M) values for UGT1A6 and UGT2B13 in SF-9 and COS-1 cells were similar. Overall, SF-9 cells seem to serve as an efficient expression system for the production of the mammalian UGTs.
Nguyen
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Department of Pharmacology, University of California-San Diego, Cancer Center, La Jolla 92093-0636, USA.
Tukey
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RH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D018076:DNA, Complementary; D015967:Gene Expression Regulation, Viral; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D018411:Spodoptera / Q000821:virology; D013997:Time Factors
D018076:DNA, Complementary; D014453:Glucuronosyltransferase