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 fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT 1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell 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 of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.
Forsman
T
T
Orion Pharma, Molecular Biology and Target Protein Research, University of Helsinki, Finland. tarja.forsman@helsinki.fi
Lautala
P
P
Lundström
K
K
Monastyrskaia
K
K
Ouzzine
M
M
Burchell
B
B
Taskinen
J
J
Ulmanen
I
I
D000818:Animals; D016466:CHO Cells / Q000201:enzymology / Q000821:virology; D002396:Catechols / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D005347:Fibroblasts / Q000201:enzymology / Q000821:virology; D015870:Gene Expression; D005822:Genetic Vectors; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000201:enzymology / Q000821:virology; D008168:Lung / Q000201:enzymology / Q000821:virology; D009570:Nitriles; D009596:Nitrophenols / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012672:Semliki forest virus / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014162:Transfection; D000090264:UDP-Glucuronosyltransferase 1A9
D002396:Catechols; D017931:DNA Primers; D009570:Nitriles; D009596:Nitrophenols; D012333:RNA, Messenger; D011994:Recombinant Proteins; C000715591:UGT1A9 protein, human; C071192:entacapone; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9; C024836:4-nitrophenol