Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT-1 cells cultured in serum-free medium.
The inability to sialylate recombinant glycoproteins is a critical limitation of the baculovirus-insect cell expression system. This limitation is due, at least in part, to the absence of detectable sialyltransferase activities and CMP-sialic acids in the insect cell lines routinely used as hosts in this system. SfSWT-1 is a transgenic insect cell line encoding five mammalian glycosyltransferases, including sialyltransferases, which can contribute to sialylation of recombinant glycoproteins expressed by baculovirus vectors. However, sialylation of recombinant glycoproteins requires culturing SfSWT-1 cells in the presence of fetal bovine serum or another exogenous source of sialic acid. To eliminate this requirement and extend the utility of SfSWT-1 cells, we have isolated a new baculovirus vector, AcSWT-7B, designed to express two mammalian enzymes that can convert N-acetylmannosamine to CMP-sialic acid during the early phase of infection. AcSWT-7B was also designed to express a model recombinant glycoprotein during the very late phase of infection. Characterization of this new baculovirus vector showed that it induced high levels of intracellular CMP-sialic acid and sialylation of the recombinant N-glycoprotein upon infection of SfSWT-1 cells cultured in serum-free medium supplemented with N-acetylmannosamine. In addition, co-infection of SfSWT-1 cells with AcSWT-7B plus a conventional baculovirus vector encoding human tissue plasminogen activator resulted in sialylation of this recombinant N-glycoprotein under the same culture conditions. These results demonstrate that AcSWT-7B can be used in two different ways to support recombinant N-glycoprotein sialylation by SfSWT-1 cells in serum-free medium. Thus, AcSWT-7B can be used to extend the utility of this previously described transgenic insect cell line for recombinant sialoglycoprotein production.
Hill
Daniel R
DR
Department of Molecular Biology, 1000 E. University Ave., University of Wyoming, Laramie, Wyoming 82071, USA.
Aumiller
Jared J
JJ
Shi
Xianzong
X
Jarvis
Donald L
DL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism*
D016895:Culture Media, Serum-Free; D006023:Glycoproteins; D011994:Recombinant Proteins; D012794:Sialic Acids