Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27
Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27
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DOI：
10.1021/bp980062h
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发表时间：
1998-09-01
影响因子：
2.9
通讯作者：
Bailey, JE
中科院分区：
文献类型：
--
作者：
Mazur, X;Fussenegger, M;Bailey, JE
我们构建了稳定的中国仓鼠卵巢（CHO）细胞系，有条件地和协调地表达模型产物基因分泌碱性磷酸酶（SEAP）和细胞生长抑制基因p21，p27，和p53175 P，p53突变缺陷的细胞凋亡，但不是细胞周期阻滞功能。双顺反子表达技术的使用允许在四环素应答启动子PhCMV*-1的控制下，以协调的方式从单个表达单元条件表达模型产物基因和细胞生长抑制基因。由于在多顺反子表达单元中存在细胞生长抑制基因，工程化CHO细胞系的生长行为可以通过向细胞培养基中添加或从细胞培养基中去除外源性试剂四环素来控制。四环素的撤回导致稳定细胞系的持续生长停滞持续较长时间。发现这种细胞系的生长停滞伴随着每个细胞的SEAP产量增加10-15倍。这种受控增殖技术允许设计新的两阶段生产方法，其由导致所需细胞密度的增殖阶段，随后是延长的生产阶段组成，在此期间细胞保持生长停滞并增加异源蛋白的细胞特异性生产。
We constructed stable Chinese hamster ovary (CHO) cell lines which conditionally and coordinately express the model product gene secreted alkaline phosphatase (SEAP) and one of the cytostatic genes p21, p27, and p53175P, a p53 mutant deficient in apoptotic but not cell-cycle arrest function. The use of dicistronic expression technology allowed the conditional expression of the model product gene and the cytostatic gene in a coordinated fashion from a single expression unit under the control of the tetracycline-responsive promoter PhCMV*-1. Due to the presence of a cytostatic gene in the multicistronic expression unit, the growth behavior of the engineered CHO cell lines could be controlled by the addition or withdrawal of the exogenous agent tetracycline to or from the cell culture medium. Withdrawal of tetracycline resulted in sustained growth arrest of the stable cell lines for a prolonged period. The growth arrest of such cell lines was found to be accompanied by a 10-15-fold increase in their production of SEAP per cell. This controlled proliferation technology allows the design of a novel two-stage production process which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain growth-arrested and increase cell-specific production of a heterologous protein.