Laboratory of Animal Cell Biotechnology, Faculty of Sciences, Université Libre de Bruxelles, CP160/17, 50 Av. F.D. Roosevelt, 1050, Brussels, Belgium,
Cytotechnology. 2001 Jul;36(1-3):71-83. doi: 10.1023/A:1014088919546.
Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 degrees C to 32 degrees C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 degrees C, at 32 degrees C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA.
通过改变物理化学条件（存在丁酸钠和温度的改变）来直接控制细胞代谢，从而调节 SV40 启动子控制下在 CHO 细胞系中表达的人重组组织型纤溶酶原激活物（t-PA）的产率。我们表明，通过添加丁酸钠或从 37°C 降低至 32°C，t-PA 的分泌量都会增加，而细胞生长则显著减少。这些处理还增加了细胞内 t-PA 的含量。我们通过细胞计数法测量细胞周期各阶段的分布，并使用 Kromenaker 和 Srienc（1991、1994a、b）的公式修改版来分析不同细胞周期阶段的细胞内 t-PA 产生率。在所有条件下（37°C、32°C 和存在丁酸钠时），细胞内 t-PA 都在 G1 期积累。此外，我们还表明，用丁酸钠处理的细胞在 G1 期的分布时间显著增加。用凝胶酶谱法和 Western blot 分析在不同细胞培养条件下产生的 t-PA：丁酸钠既没有改变蛋白质的整体质量，也没有改变温度的变化。还分析了用碳水化合物特异性凝集素分析重组人 t-PA 的 N-糖基化模式。丁酸钠引起 N-连接的复杂高甘露糖寡糖的短暂增加，而对 t-PA 的唾液酸含量没有影响。
