Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis
Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis
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DOI：
10.1023/a:1014088919546
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发表时间：
2001-01-01
期刊：
影响因子：
2.2
通讯作者：
Werenne, J
中科院分区：
文献类型：
--
作者：
Hendrick, V;Winnepenninckx, P;Werenne, J
在中国仓鼠卵巢(CHO)细胞系中，通过改变理化条件(丁酸钠的存在和温度的改变)直接控制细胞代谢来调节SV40启动子控制下表达的重组人组织型纤溶酶原激活剂(t-PA)的产量。我们发现，无论是加入丁酸钠还是将温度从37℃降至32℃，t-PA的排泄量都会增加，而细胞的生长却显著减少。用流式细胞仪检测细胞周期时相的分布，并用修正的Kromenaker和Srienc(1991,1994 a，b)公式分析不同细胞周期时相细胞内t-PA的生成率。细胞内t-PA在所有条件下(37℃、32℃和丁酸盐存在时)均在G1期积聚。此外，我们还发现，经丁酸处理的TUNE细胞的分布明显增加，维持在G1期细胞周期。对所测试的不同细胞培养条件下产生的t-PA进行了酶谱和Western blotting分析：无论是丁酸，还是温度的变化都没有显著改变蛋白质的整体质量。用糖类特异性凝集素分析重组人t-PA的N-糖链模式。丁酸对t-PA的唾液酸含量无影响，但使N-连接复合体高甘露糖寡糖含量短暂增加。
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 37degreesC to 32degreesC 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 G 1 phase in all conditions (at 37degreesC, at 32degreesC and in presence of butyrate). Moreover, we have shown that the distribution of the tune cells treated by butyrate are maintained in the G1 cell 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.