Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells.
An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase-encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N-glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.
Hollister
J R
JR
Department of Entomology and Center for Advanced Invertebrate Molecular Sciences, Texas A&M University 77843, USA.
Shaper
J H
JH
Jarvis
D L
DL
D000818:Animals; D016367:Baculoviridae / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D008322:Mammals; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D014779:Virus Replication
D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase; D010959:Tissue Plasminogen Activator