Abstract
We have previously reported that Namalwa KJM-1 cells adapted to a serum-free medium and derived from Namalwa (B lymphoblastoid) cells were more useful than CHO (Chinese hamster ovary) cells as the host cell line for producing various recombinant proteins with a dhfr gene coamplification method, because CHO cells secrete a cysteine endopeptidase (Satoh, 1990).
An expression plasmid for pro-UK was constructed and introduced into CHO and Namalwa KJM-1 cells, and cells resistant to methotrexate (MTX) were then obtained. A Western blot analysis of pro-UK produced by Namalwa KJM-1 showed a single chain form, without the addition of protease inhibitors. However, pro-UK produced by CHO was partly cleaved to a two-chain form that was not activatable by plasmin, even with the addition of Aprotinin (10KIU/ml).
Pro-UKS1 was designed as a thrombin-resistant derivative of pro-UK by introducing sugar chains, Phe being substituted with Asn at the 164th amino acid residue of pro-UK. An expression plasmid for pro-UKS1 was constructed and introduced into Namalwa KJM-1 cells, and cells resistant to MTX were then obtained. Among them, the highest pro-UKS1 producer (resistant to 500nM of MTX), clone 41–8, was selected and further characterized. Clone 41–8 was cultured in a serum-free (ITPSGF) medium, and under conventional conditions, the maximal productivity of pro-UKS1 was about 10µg/ml/day. The addition of glucose and tri-iodothyronine was effective for increasing the productivity, the maximal productivity of pro-UKS1 being 67µg/ml/day. In this conditioned medium, the content of pro-UKS1 was above 80% of the total protein.
These results indicate that Namalwa KJM-1 cells are a useful host cell line for producing recombinant proteins susceptible to proteases.
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© 1993 Springer Science+Business Media Dordrecht
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Hosoi, S. et al. (1993). Stable Production of Pro-urokinase and its Derivative by Namalwa KJM-1 Cells Adapted to a Serum-free Medium. In: Kaminogawa, S., Ametani, A., Hachimura, S. (eds) Animal Cell Technology: Basic & Applied Aspects. Animal Cell Technology: Basic & Applied Aspects, vol 5. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-2044-9_9
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DOI: https://doi.org/10.1007/978-94-011-2044-9_9
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