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