Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UK delta GS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium.

Pro-UK delta GS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UK delta GS1SEd1-5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UK delta GS1 producer (resistant to 200 nM of MTX), clone 2-9, was selected and used for further studies. Under the conventional conditions, i.e. at 37 degrees C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UK delta GS1 produced by clone 2-9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UK delta GS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri- and tetraantennary complex-type oligosaccharide. The modulated pro-UK delta GS1 showed superior in vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.