High-level scu-PA production by butyrate-treated serum-free culture of recombinant CHO cell line.
The MGpUK-5 cell line, transformed with a single-chain urokinase-type plasminogen activator (scu-PA) minigene, generated mRNA transcripts and scu-PA titers corresponding to 65% or 86% of the amount generated before serum-free adaptation, despite significant loss of scu-PA gene copies during adaptation to serum-free culture. To further augment scu-PA production, a culture strategy employing sodium butyrate was explored. In 60-mL spinner flask cultures, sodium butyrate in the concentration range 1-10 mM allowed scu-PA production 2- to 3-fold higher than that in the negative control culture. Its productivity-enhancing activity was dependent on cell density in a range of 1-5 x 10(6) cells/mL, generating 72,200 +/- 8,100 IU/mL (480 +/- 50 mg/L) in 60-mL spinner flask cultures. To confirm this result, cells were grown to 4.4 x 10(6) cells/mL and treated with 5 mM sodium butyrate in a 2.5-L perfusion culture. The scu-PA titer increased more than 2-fold, and specific production rate of scu-PA increased 3-fold by this treatment. Overall, this perfusion culture gave rise to 1.7 x 10(8) IU scu-PA (1.1 g), comparable to total scu-PA production in a batch butyrate-treated culture performed at a 25-L bioreactor scale (1.3-3.5 g). Our results suggest that sodium butyrate treatment on high-density culture enables scu-PA production in gram quantities.
Kim
Jung-Seob
JS
MOGAM Biotechnology Research Institute, 341 Pojung-Ri, Koosung-Eup, Yongin, Kyonggi-Do 449-913, Republic of Korea.
Ahn
Byung-Chul
BC
Lim
Byung-Pil
BP
Choi
Yang Do
YD
Jo
Eui-Cheol
EC
D000818:Animals; D002087:Butyrates / Q000008:administration & dosage; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D002087:Butyrates; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator