Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1).
Human uridinediphosphate-glucuronosyltransferase 1A1 (UGT1A1) was expressed in Salmonella typhimurium TA1535 cells by transfection of the cells with plasmids carrying the UGT1A1 cDNA. UGT1A1 cDNA was isolated by a polymerase chain reaction from human liver total RNA and was inserted into the pSE420 plasmid, linked to the trc promoter and terminator. The plasmid thus constructed was introduced into Salmonella TA1535 cells. The expression of human UGT1A1 protein was confirmed by Western blot analysis. The maximal expression was observed at 24 h after the addition of isopropyl-beta-D-thiogalactopyranoside, an inducer. However, the bilirubin conjugation activity of the membrane fraction from the Salmonella cells was not detectable. When a beta-glucuronidase inhibitor such as saccharic acid 1,4-lactone, glycyrrhizin or 1-naphtyl-beta-D-glucuronide was added to the reaction mixture, the bilirubin conjugation activity of the human UGT1A1 was detected. When geniposide was added to the reaction mixture, the bilirubin conjugation activity of UGT1A1 was not seen. Taking these results into account, the established Salmonella strain possesses the beta-glucuronidase activity. Since the beta-glucuronidase activity of the Salmonella was lower than that of E. coli, it was concluded that Salmonella seemed to be a good host to express UGT protein. This is the first study to demonstrate the establishment of a bacterial strain expressing native human UGT protein showing catalytic activity.
Fujita
K
K
Laboratory of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Mogami
A
A
Hayashi
A
A
Kamataki
T
T
D000818:Animals; D001663:Bilirubin / Q000378:metabolism; D015153:Blotting, Western; D002458:Cell Fractionation; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005419:Flavonoids / Q000378:metabolism; D015870:Gene Expression; D005965:Glucuronates / Q000494:pharmacology; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019695:Glycyrrhizic Acid / Q000494:pharmacology; D006801:Humans; D039823:Iridoids; D008862:Microsomes, Liver / Q000201:enzymology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011714:Pyrans / Q000494:pharmacology; D012313:RNA / Q000032:analysis; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005965:Glucuronates; D039823:Iridoids; D011714:Pyrans; C007835:geniposide; C038044:baicalin; D012313:RNA; D019695:Glycyrrhizic Acid; C029091:naphthyl glucuronide; D014453:Glucuronosyltransferase; D005966:Glucuronidase; D001663:Bilirubin