Mild hypothermia improves transient gene expression yields several fold in chinese hamster ovary cells
Mild hypothermia improves transient gene expression yields several fold in chinese hamster ovary cells
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DOI：
10.1021/bp070286c
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发表时间：
2008-03-01
影响因子：
2.9
通讯作者：
Wurm, Florian M.
中科院分区：
文献类型：
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作者：
Wulhfard, Sarah;Tissot, Stephanie;Wurm, Florian M.
哺乳动物细胞中的大规模瞬时基因表达（TGE）是产生重组蛋白的快速方法，但是分泌蛋白的体积生产率仍然比通常用重组细胞系实现的产量低一个数量级以上。在此，在用线性25 kDa聚乙烯亚胺转染的中国仓鼠卵巢细胞中瞬时重组蛋白的产生通过在DNA递送后在29至33 ℃的温度下孵育细胞而显著增强。用这种方法，在转染的6天内实现了60-80 mg/L的瞬时重组抗体产量。TGE的增加与细胞在细胞周期的G1期的积累、细胞大小的增加、细胞活力的提高、转基因mRNA的稳态水平的提高、营养物质的消耗的减少和废物的积累的减少相关。TGE的增强不是载体依赖性的，但是在转基因mRNA的3'非翻译区中土拨鼠肝炎病毒转录后调节元件的存在使瞬时重组抗体表达在31 ℃下比在37 ℃下的表达增加超过3倍。通过低温增强CHO细胞中的TGE实现的产率使得该技术对于大规模（高达100 L）快速生产克量的分泌重组蛋白是可行的。
Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 degrees C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3' untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31 degrees C as compared to expression at 37 degrees C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).