title: Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells.
abstract: When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Sung
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D020148:Butyric Acid / Q000494:pharmacology*; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D046508:Culture Techniques / Q000379:methods; D004305:Dose-Response Relationship, Drug; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects; D012794:Sialic Acids / Q000378:metabolism; D013535:Suspensions; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000187:drug effects; D013997:Time Factors; D014162:Transfection
chemical_list: D016895:Culture Media, Serum-Free; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012794:Sialic Acids; D013535:Suspensions; D020148:Butyric Acid; D013926:Thrombopoietin; D007770:L-Lactate Dehydrogenase; D006026:Glycoside Hydrolases