Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme.
Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.
Kurkela
Mika
M
Viikki Drug Discovery Technology Center, University of Helsinki, Finland.
García-Horsman
J Arturo
JA
Luukkanen
Leena
L
Mörsky
Saila
S
Taskinen
Jyrki
J
Baumann
Marc
M
Kostiainen
Risto
R
Hirvonen
Jouni
J
Finel
Moshe
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA; D003902:Detergents / Q000494:pharmacology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003902:Detergents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D004247:DNA; D014453:Glucuronosyltransferase