Makrydaki Elli, Marshall Oscar, Heide Chiara, Buldum Gizem, Kontoravdi Cleo, Polizzi Karen M
Department of Chemical Engineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, United Kingdom.
Department of Chemical Engineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, United Kingdom.
Methods Enzymol. 2021;659:411-435. doi: 10.1016/bs.mie.2021.08.004. Epub 2021 Sep 15.
Cell-free protein synthesis (CFPS) platforms can be used for rapid and flexible expression of proteins. The use of CFPS platforms from mammalian, specifically Chinese hamster ovary (CHO) cells, offers the possibility of a rapid prototyping platform for recombinant protein production with the capabilities of post-translational modifications. In this chapter, we discuss a refined CFPS system based on CHO cells, including: extract preparation, reaction mix composition, and accessory protein supplementation to enhance expression. Specifically, when the CHO cell extract is combined with a truncated version of GADD34 and K3L, stress-induced eIF2 phosphorylation is reduced and inhibition of translation initiation is relieved, increasing yields. A brief summary of the protocol for running the CFPS reactions is also described. Overall, the method is reliable and leads to a highly reproducible expression system. Finally, the advantages and disadvantages of the platform, in addition to expected outcomes, are also discussed.
无细胞蛋白质合成（CFPS）平台可用于蛋白质的快速灵活表达。使用来自哺乳动物，特别是中国仓鼠卵巢（CHO）细胞的CFPS平台，为具有翻译后修饰能力的重组蛋白生产提供了一个快速原型平台。在本章中，我们讨论了一种基于CHO细胞的优化CFPS系统，包括：提取物制备、反应混合物组成以及补充辅助蛋白以增强表达。具体而言，当CHO细胞提取物与截短版的GADD34和K3L结合时，应激诱导的eIF2磷酸化减少，翻译起始抑制得到缓解，产量增加。还描述了进行CFPS反应的方案的简要总结。总体而言，该方法可靠，可导致高度可重复的表达系统。最后，还讨论了该平台的优缺点以及预期结果。
