Department of Chemical Engineering, University of Colorado, 80309-0424, Boulder, CO, USA.
Cytotechnology. 1996 Jan;22(1-3):179-84. doi: 10.1007/BF00353937.
Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular β-galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular β-galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.
已发现，常用的 SV40 启动子的外源蛋白表达主要发生在细胞周期的 S 期。具有这种细胞周期依赖性外源蛋白表达的简单数学模型表明，特定的生产速率将与细胞生长速率成正比，这在高细胞密度补料分批或灌注生物反应器中特别不利。在这项研究中，我们通过在连续悬浮生物反应器中培养重组 CHO 细胞来研究生产速率和生长速率之间的这种预测关系。一种 CHO 细胞系 GS-26 已通过质粒 pSVgal 稳定转染，该质粒在 SV40 启动子的控制下包含大肠杆菌 lacZ 基因。该 GS-26 细胞系在分批和连续模式下在一系列特定生长速率下在悬浮培养物中生长。在打破细胞并释放酶后，使用标准分光光度法测定细胞内β-半乳糖苷酶活性。正如预测的那样，在分批和连续培养中，发现细胞内β-半乳糖苷酶含量与比生长速率之间存在很强的生长相关性。
