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Research ArticleArticle

Baculovirus-Directed Expression of Rabbit UDP-Glucuronosyltransferases in Spodoptera frugiperda Cells

Nghia Nguyen and Robert H. Tukey
Drug Metabolism and Disposition June 1997, 25 (6) 745-749;
Nghia Nguyen
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Robert H. Tukey
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Abstract

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 ∼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,KM 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.

Footnotes

  • Send reprint requests to: Dr. Robert H. Tukey, Department of Pharmacology, University of California–San Diego, Cancer Center, La Jolla, CA 92093–0636.

  • This study was supported by U.S. Public Health Service Grant GM49135.

  • Abbreviations used are::
    UGT
    UDP-glucuronosyltransferase
    UDPGA
    UDP-glucuronic acid
    SF-9
    Spodopera frugiperda
    UGT2B13
    4-hydroxybiphenyl UGT
    UGT1A6
    previously characterized as UGT1.6 (8) (based upon a recommended nomenclature at the recent “VIIIth International Workshop on Glucuronidation and UDP-Glucuronosyltransferases” held in Iowa City, IA, May 19–22, 1996
    the designation of UGT1.6 is now UGT1A6)
    IgG, immunoglobulin G
    dNTP
    deoxy nucleotide triphosphate
    AcMNPV
    Autographa californica nuclear polyhedrosis virus
    PBS
    phosphate-buffered saline
    SDS
    sodium dodecyl sulfate
    blocking buffer
    PBS containing 0.1% Tween-20 and 5% carnation nonfat dry milk
    RIPA buffer
    50 mM Tris-HC1 (pH 7.5) containing 10% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 150 mM NaCl, and 1 mM phenylmethanesulfonyl fluoride
    SDS-PAGE
    SDS-polyacrylamide gel electrophoresis
    • Received October 17, 1996.
    • Accepted February 20, 1997.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition
Vol. 25, Issue 6
1 Jun 1997
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Research ArticleArticle

Baculovirus-Directed Expression of Rabbit UDP-Glucuronosyltransferases in Spodoptera frugiperda Cells

Nghia Nguyen and Robert H. Tukey
Drug Metabolism and Disposition June 1, 1997, 25 (6) 745-749;

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Research ArticleArticle

Baculovirus-Directed Expression of Rabbit UDP-Glucuronosyltransferases in Spodoptera frugiperda Cells

Nghia Nguyen and Robert H. Tukey
Drug Metabolism and Disposition June 1, 1997, 25 (6) 745-749;
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