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.

中文翻译：

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高效的人类无细胞翻译系统

无细胞蛋白质合成 (CFPS) 系统可轻松实现蛋白质的体外表达，并具有许多科学、工业和治疗应用。在这里，我们提出了一种优化的、高效的人类无细胞翻译系统，它绕过了当前使用的体外系统的许多限制。该 CFPS 系统基于人 HEK293T 细胞的提取物，该细胞被设计为内源性表达 GADD34 和 K3L 蛋白，抑制翻译起始因子 eIF2α 的磷酸化。在细胞裂解物制备之前在人细胞中过度表达 GADD34 和 K3L 蛋白可显着简化裂解物的制备。我们发现细胞裂解前 GADD34 和 K3L 辅助蛋白的表达维持了提取物中 eIF2α 的低磷酸化水平。在体外翻译反应期间，eIF2α 磷酸化以 GCN2 依赖性方式适度增加，可被 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。