Beta-(1 --> 4)-galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence.
To evaluate the ability of insect cells to produce complex-type N-glycans, beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity in several insect cell lines was analyzed. For this purpose, we developed a simple and highly sensitive assay for beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity, which is based on time-resolved fluorometry of europium. Bovine serum albumin (BSA) modified with GlcNAc (GlcNAc(44)-BSA) was used as the acceptor. GlcNAc(44)-BSA was coated on a 96-well microplate, and after incubation with the enzyme sample in the presence of UDP-Gal, Eu-labeled RCA(120) (Ricinus communis aggutin I), was added. RCA(120) binds to the Galbeta(1 --> 4)GlcNAc structure in the product, and the bound Eu-RCA(120) was measured by the fluorescence of europium. When bovine beta4Gal-T-I was used as a standard reference enzyme, a linear relationship between enzyme activity and fluorescent signal was obtained over the range of 0-1000 microUnits (IU). Using this system, we were able to measure a low but significant level of beta4GalT activity in Trichoplusia ni cells ('High Five'). In contrast, no endogenous beta4GalT activity was detected in a Spodoptera frugiperda (Sf-9) cell line. However, Sf-9 cells stably transfected with the bovine beta4GalT-I gene and 'High Five' cells infected with a baculovirus containing the same gene produced activity levels that were comparable to or greater than those found in Chinese hamster ovary cells. We also showed that the beta4GalT activity level observed in the baculovirus-infected T. ni cells under the control of immediate early promoter was highly dependent on the post-infection time, suggesting that galactosylation level may also be variable during the infection period.
Abdul-Rahman
Badarulhisam
B
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Ailor
Erik
E
Jarvis
Donald
D
Betenbaugh
Michael
M
Lee
Yuan Chuan
YC
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D005063:Europium; D005470:Fluorometry; D007313:Insecta / Q000166:cytology*; D007700:Kinetics; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis; D014162:Transfection
D011134:Polysaccharides; D005063:Europium; D009237:N-Acetyllactosamine Synthase