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.

MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells

“…In general most researchers have observed increased maximum cell number, enhanced viability, extended culture duration, protection from viral and sodium butyrate mediated apoptosis and in certain cases increased titers following expression of Bcl family members. (Fassnacht et al 1999;Tey et al 2000;Goswami et al 1999;Simpson et al 1999;Fussenegger et al 2000;Mastrangelo et al 2000a, b;Meents et al 2002;Chiang and Sisk 2005;Kim and Lee 2002;Sung and Lee 2005;Sauerwald et al 2006;Ifandi and Al-Rubeai 2005). Viral homologues of Bcl-2 including E1B19K have also been used for the inhibition of apoptosis in mammalian cell cultures (Wong et al 2006;Figueroa et al 2006), where E1B19K inhibits the apoptotic activity of p53 (Debbas and White 1993;Lowe and Ruley 1993).…”

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

“…Addition of sodium butyrate (NaBu) has also been used as an efficient method to enhance specific protein productivity (q) in CHO cells (Chang et al, 1999;Kim and Lee, 2000;Laubach et al, 1996;Palermo et al, 1991;Sung and Lee, 2005;Sung et al, 2004). NaBu, a short-chain fatty acid, inhibits the activity of histone deacetylase, and the hyperacetylation of histone proteins induces more access of general transcription factors to DNA (Kruh, 1982;Lee et al, 1993;Riggs et al, 1977).…”

“…For example, overexpression of members of the Bcl-2 family including Bcl-2 and Bcl-x L has been employed to prevent apoptotic cell death induced by various causes such as addition of NaBu (Kim and Lee, 2000;Sung and Lee, 2005;Wang et al, 2004), hyperosmolarity (Kim and Lee, 2002), nutrient depletion (Tey et al, 2000), and low culture temperature (Kim and Lee, 2009). However, the intracellular portraits of the CHO cell proteome in response to Bcl-x L overexpression are still poorly understood despite the number of genetic engineering studies that have been performed.…”

Anti‐apoptotic genes Aven and E1B‐19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture

“…These studies have explored the use of anti-apoptotic genes from the Bcl-2 family (Arden and Betenbaugh, 2004;Figueroa et al, 2001;Goswami et al, 1999;Meents et al, 2002;Sauerwald et al, 2006;Singh et al, 1996;Sung and Lee, 2005;Tey et al, 2000a,b). Others have explored the use of functional viral homologs of Bcl-2, such as the adenovirus E1B-19K protein , the Kaposi sarcoma-associated virus KSBcl-2 protein and the Epstein-Barr virus BHRF-1 protein (Vives et al, 2003).…”