Abstract
Pro-UKΔGS1 was designed as a stable and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site at near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKΔGS1, plH1UKΔGS1SEd1-5, was constructed using dhfr and pSVneo selectable markers and introduced into Namalwa KJM-1 cells. Cells resistance to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKΔGS1 producer (resistant to 200nM MTX), clone 2–9, was selected and used for further studies.
Under conventional culture conditions, the oligosaccharide structure of pro-UKΔGS1 produced by Namalwa KJM-1 cells adapted to a serum-free medium mainly consisted fucose (Fuc)-containing biantennary complex-type oligosaccharides. Addition of dexamethasone (Dex), changing carbohydrate contents of the media, and shift down of incubation temperature (temp) changed oligosaccharide structure of pro-UKΔGS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri- and tetraantennary complex-type oligosaccharides. The modified pro-UKΔGS1 showed superior in vivo activity against a canine femoral thrombosis.
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Hosoi, S. et al. (1994). Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by culture conditions. In: Kobayashi, T., Kitagawa, Y., Okumura, K. (eds) Animal Cell Technology: Basic & Applied Aspects. The Sixth International Meeting of Japanese Association for Animal Cell Technology JAACT’93, vol 6. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-0848-5_35
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DOI: https://doi.org/10.1007/978-94-011-0848-5_35
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