Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability |
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Authors: | Mitsuo Satoh Shinji Hosoi Hiromasa Miyaji Seiga Itoh Seiji Sato |
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Institution: | (1) Tokyo Research Laboratories, Kyowa Hakko Co., Ltd., 3-6-6 Asahi-machi, 194 Machida-shi, Tokyo, Japan |
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Abstract: | Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/106 cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS
2,2 -azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt
- AMC
7-amino-4-methylcoumarin
- CHO
Chinese hamster ovary
- DFP
diisopropylfluorophosphate
- DHFR
dihydrofolate reductase
- ELISA
enzyme-linked immunosorbent assay
- MCA
4-methylcoumaryl-7-amide
- MTX
methotrexate
- NEAA
non essential amino acid
- NEM
N-ethylmaleimide
- PCMB
p-chloromercuribenzonate
- PMSF
phenylmethanesulfonyl fluoride
- pro-UK
pro-urokinase |
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Keywords: | Chinese hamster ovary cells Namalwa KJM-1 cells protease activity pro-urokinase serum-free medium stability of expressed-protein |
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