Sung Yun Hee, Song Yeon Jung, Lim Seung Wook, Chung Joo Young, Lee Gyun Min
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, South Korea.
J Biotechnol. 2004 Sep 9;112(3):323-35. doi: 10.1016/j.jbiotec.2004.05.003.
Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.
人血小板生成素（hTPO）是一种高度糖基化的蛋白质，分别具有6个和24个潜在的N-糖基化位点和O-糖基化位点。为了确定丁酸钠（NaBu）对重组中国仓鼠卵巢（rCHO）细胞中hTPO产生及质量的影响，将NaBu（0 - 10 mM）添加到指数生长期细胞的培养物中。添加NaBu显著提高了hTPO的比产量和体积产量，尽管它以剂量依赖的方式通过凋亡降低了细胞活力。在添加3 mM NaBu的培养物中获得了最高hTPO浓度，为82.2±5.6 μg/ml-1。与未添加NaBu的培养物相比，添加3 mM NaBu的培养物在第7天导致qTPO增加6.4倍，最终hTPO浓度增加3.3倍。然而，NaBu使hTPO的质量恶化，这是由于异质性增加、酸性hTPO亚型减少、α（2→3）唾液酸化减少以及体内生物活性降低所致。我们还发现，第7天收集的添加3 mM NaBu的培养物中hTPO的生物活性是未添加NaBu的培养物中hTPO生物活性的72%。综上所述，对于像hTPO这样的高度糖基化蛋白质的高水平表达，使用NaBu或其最佳浓度应考虑其对糖蛋白质量的有害影响来确定。
