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Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT‐1 cells cultured in serum‐free medium
Author(s) -
Hill Daniel R.,
Aumiller Jared J.,
Shi Xianzong,
Jarvis Donald L.
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20945
Subject(s) - recombinant dna , glycoprotein , sialic acid , cell culture , biology , biochemistry , microbiology and biotechnology , gene , genetics
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. © 2006 Wiley Periodicals, Inc.