Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian beta1,4-galactosyltransferase.
Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine beta-1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.
Wolff
M W
MW
Department of Chemical and Biochemical Engineering, University of Wyoming, Laramie 82071, USA.
Murhammer
D W
DW
Jarvis
D L
DL
Linhardt
R J
RJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006001:Glycoconjugates / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D006001:Glycoconjugates; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase