Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium |
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Authors: | Shinji Hosoi Mitsuo Satoh Hiromasa Miyaji Tatsunari Nishi Tamio Mizukami Mamoru Hasegawa Seiga Itoh Tatsuya tamaoki |
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Affiliation: | (1) Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, 194 Machida-shi, Tokyo, Japan |
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Abstract: | Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml–1. Addition of glucose and tri-iodothyronine (T3) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml–1 day–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T3 tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80 |
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Keywords: | Namalwa KJM-1 cells pro-urokinase thrombin resistant pro-UKS1 stable production culture conditions serum-free medium |
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