Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the Semliki Forest virus expression system

Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibro blast V79 cells with recombinant SFV-UGT viruses resulted in efficient prot ein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 c ell line. No UGT signal was detected in noninfected cells. In addition, SFV -UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4 ) cells leading to high production of recombinant enzyme. The measurement o f enzyme activities and kinetic parameters using p-nitrophenol and nitrocat echol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showe d that the overall kinetic properties of the enzymes produced by the two sy stems were similar. We conclude that the SFV expression system represents a n efficient, fast and versatile method for production of metabolic enzymes for in vitro assays. (C) 2000 Elsevier Science Inc. All rights reserved.