A highly efficient human cell-free translation system.
A highly efficient human cell-free translation system.
复制标题
高效的人类无细胞翻译系统。
DOI：
10.1101/2023.02.09.527910
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Cate,JamieHD
中科院分区：
文献类型：
--
作者：
Aleksashin,NikolayA;Chang,StaceyTsai-Lan;Cate,JamieHD
无细胞蛋白合成（CFPS）系统使许多科学，工业和治疗应用的蛋白质易于体外表达。在这里，我们提出了一个优化的，高效的人类无细胞翻译系统，绕过了目前在体外系统中使用的许多限制。该CFPS系统基于人HEK293T细胞提取物，内源性表达GADD34和K3L蛋白，抑制翻译起始因子eIF2α的磷酸化。在细胞裂解液制备前，在人细胞中过表达GADD34和K3L蛋白可显著简化裂解液制备。我们发现，在细胞裂解前，GADD34和K3L辅助蛋白的表达维持了提取物中eIF2α的低水平磷酸化。在体外翻译反应中，eIF2α磷酸化以GCN2依赖性的方式适度增加，可以被GCN2激酶抑制剂抑制。这种新的CFPS系统应该有助于探索细胞外更多生理条件下的人类翻译机制。
Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.