Human UDP-glucuronosyltransferase expression in insect cells: ratio of active to inactive recombinant proteins and the effects of a C-terminal his-tag on glucuronidation kinetics.
Many laboratories use recombinant UDP-glucuronosyltransferases (UGTs), expressed in baculovirus-infected insect cells, for drug glucuronidation studies. We have infected Sf9 insect cells with increasing amounts of recombinant baculovirus, encoding either UGT1A9 or UGT2B7, and measured both glucuronidation activity and immunodetectable UGT in the resulting cell homogenates. The correlation between glucuronidation rates and degree of infection followed different trends, depending on whether activity was the actual activity measured or was corrected for UGT expression level. Above a certain low level of infection, further increases in infection ratios led to a large decline in normalized activity, presumably due to the presence of full-length but inactive enzyme in the sample. Because immunodetection does not distinguish between active and inactive UGT, comparison of normalized activity between different batches of a recombinant UGT, mutants of a given UGT, or different UGTs is prone to large inaccuracies. Such inaccuracies could be reduced by lowering the degree of infection of the insect cells, in combination with careful monitoring of UGT expression. However, the latter requires suitable antibodies for comparing UGT expression levels among preparations, antibodies that are not always available. Poly-His (His-tag)-containing peptides, fused to the UGT C terminus, allow sensitive immunodetection of expressed enzymes with monoclonal antibodies. We have now carefully examined the effects of two such fusion peptides on enzyme kinetics. A minor increase in the K(m) values has been detected in the His-tagged UGTs, but no changes in parameters such as the kinetic model and the effects of albumin addition.
Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Finland.
Hongbo
H
Zhang
Anne-Sisko
AS
Patana
Peter I
PI
Mackenzie
Shinichi
S
Ikushiro
Adrian
A
Goldman
Moshe
M
Finel
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D020134:Catalytic Domain; D004789:Enzyme Activation; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D000090264:UDP-Glucuronosyltransferase 1A9
D020719:Glucuronides; D011993:Recombinant Fusion Proteins; C000715591:UGT1A9 protein, human; C033223:polyhistidine; D006639:Histidine; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9