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High-level expression and characterization of single chain urokinase-type plasminogen activator (scu-PA) produced in recombinant Chinese hamster ovary (CHO) cells

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Abstract

The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, MGpUKs, recreted a completely processed scu-PA of 54kD and up to 60 mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified as having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0 μM of MTX. Meanwhile, the transcripts of the scu-PA gene increased, although-early saturation of transcription was identified at 0.1 μM of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the large-scale production of scu-PA.

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  1. MOCAM Biotechnology Institute, 341 Pojung-Ri, Koosung-Eup, 449-913, Kyonggi-Do, Korea

    Jung-Seob Kim, Mi-Kyung Min & Eui-Cheol Jo

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Kim, JS., Min, MK. & Jo, EC. High-level expression and characterization of single chain urokinase-type plasminogen activator (scu-PA) produced in recombinant Chinese hamster ovary (CHO) cells. Biotechnol. Bioprocess Eng. 6, 117–127 (2001). https://doi.org/10.1007/BF02931957

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