Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature
Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature
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DOI：
10.1371/journal.pone.0123562
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发表时间：
2015-04-20
期刊：
影响因子：
3.7
通讯作者：
Niu, Li
中科院分区：
文献类型：
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作者：
Lin, Chi-Yen;Huang, Zhen;Niu, Li
动物细胞和细胞系，例如 HEK-293 细胞，通常在 37°C 下培养。这些细胞通常用于表达重组蛋白。具有更高的表达水平或更高的蛋白质产量通常是期望的。正如我们在本研究中所证明的，在 HEK-293S 细胞中瞬时转染 24 小时后，将培养温度降至 33 摄氏度，但不能更低，绿色荧光蛋白 (GFP) 和 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸 (AMPA) 受体的表达量将提高 1.5 倍。通过跟踪表达 GFP 的细胞从转染后 24 小时开始在 37°C 和 33°C 下生长的时间进程（包括在正常生长培养基中于 37°C 恢复 19 小时），我们发现轻度低温（即 33°C）会降低 HEK-293S 细胞的生长速率，同时增加重组蛋白的细胞生产力。结果，绿色细胞在较长时间内保持不分裂。毫不奇怪，在 33 摄氏度下生长的细胞中表达的重组蛋白的特性不受影响，如 AMPA 受体的使用所示。我们通过使用 PC12 细胞进一步证明，当使用基于化学的瞬时转染方法难以表达重组蛋白时，该方法可能特别有用。
Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37 degrees C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33 degrees C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to similar to 1.5-fold higher expression of green fluorescent protein (GFP) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37 degrees C and 33 degrees C from 24 hours after transfection (including 19 hours recovery at 37 degrees C in the normal growth medium), we found that a mild hypothermia (i.e., 33 degrees C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33 degrees C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.