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Cytotechnology

, Volume 19, Issue 2, pp 125–135 | Cite as

Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium

  • Shinji Hosoi
  • Mitsuo Satoh
  • Katsuya Higo
  • Seiji Sugimoto
  • Hiromasa Miyaji
  • Akira Karasawa
  • Kazuo Yamaguchi
  • Mamoru Hasegawa
  • Tatsuya Tamaoki
Regular Articles

Abstract

Pro-UKΔGS1 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, pIH1UKΔGS1SEd1–5 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-UKΔGS1 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°C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UKΔGS1 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-UKΔGS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri-and tetraantennary complex-type oligosaccharide. The modulated pro-UKΔGS1 showed superiorin vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.

Key words

culture conditions oligosaccharide structure modulation Namalwa KJM-1 pro-urokinase derivative 

Abbreviations

Dex

Dexamethasone

dhfr

dihydrofolate reductase

DO

dissolved oxygen

EPO

erythropoietin

Fruc

fructose

Fuc

fucose

Gal

galactose

Glu

glucose

GlcN

glucosamine

Hepes

4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid

kb

kilobase pairs

kDa

kilodalton

Man

mannose

MTX

methotrexate

pro-UK

pro-urokinase

RA

retinoic acid

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

T3

tri-iodothyronine

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Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Shinji Hosoi
    • 1
  • Mitsuo Satoh
    • 1
  • Katsuya Higo
    • 2
  • Seiji Sugimoto
    • 1
  • Hiromasa Miyaji
    • 1
  • Akira Karasawa
    • 2
  • Kazuo Yamaguchi
    • 3
  • Mamoru Hasegawa
    • 1
  • Tatsuya Tamaoki
    • 1
  1. 1.Tokyo Research LaboratoriesKyowa Hakko Kogyo Co. Ltd.Machida-shi, TokyoJapan
  2. 2.Pharmaceutical Research LaboratoriesKyowa Hakko Kogyo Co. Ltd.ShizuokaJapan
  3. 3.Pharmaceutical Research LaboratoriesKyowa Hakko Kogyo Co. Ltd.ShizuokaJapan

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