N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines |
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Authors: | P C Kulakosky M L Shuler H A Wood |
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Institution: | (1) Boyce Thompson Institute for Plant Research, Tower Road, 14853 Ithaca, New York;(2) School of Chemical Engineering, Cornell University, 14853 Ithaca, New York |
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Abstract: | Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared.
The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography.
The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate
electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose
residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides
that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The
S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose. |
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Keywords: | Trichoplusia ni Spodoptera frugiperda insect cell culture baculovirus glycoprotein alkaline phosphatase |
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