mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.
mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.
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DOI：
10.1042/bcj20160845
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发表时间：
2016-12-15
期刊：
影响因子：
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通讯作者：
Smales CM
中科院分区：
文献类型：
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作者：
Jossé L;Xie J;Proud CG;Smales CM
许多基于蛋白质的生物治疗剂在培养的中国仓鼠卵巢（CHO）细胞系中产生。最近的报道已经证明，重组mRNA的翻译和通过哺乳动物雷帕霉素靶（mTOR）信号传导对翻译机制的全局控制是这些细胞可以产生的重组蛋白质的量和质量的重要决定因素。mTOR复合物1（mTORC 1）是细胞生长/分裂、核糖体生物发生和蛋白质合成的主要调节剂，但mTORC 1信号传导、细胞生长和增殖与哺乳动物细胞的重组蛋白产量之间的关系，以及是否可以操纵该主要调节信号传导途径以增强细胞生物量和重组蛋白产量（rPP）尚未得到充分探索。我们研究了mTORC 1信号和活性，在一组表达不同量的模型单克隆IgG 4的姐妹重组谷氨酰胺合成酶-CHO细胞系的分批培养中，以评估mTORC 1信号与细胞增殖、自噬、重组蛋白表达、全局蛋白合成和mRNA翻译起始之间的联系。我们发现mTORC 1底物4 E结合蛋白1（4 E-BP 1）的表达在整个细胞培养过程中波动，正如预期的那样，4 E-BP 1磷酸化谱在整个培养过程中发生变化。重要的是，我们发现eIF 4 E/4 E-BP 1化学计量与细胞生产力正相关。此外，eIF 4 E的量似乎与4 E-BP 1的量共调节。这可能反映了在mRNA水平而不是蛋白质水平的变化的感知，或者磷酸化状态以及存在的4 E-BP 1的量在eIF 4 E和4 E-BP 1的共调节中是重要的。
Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.