Innovative Genomics Institute, University of California-Berkeley, Berkeley, California 94720, USA.
Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, California 94720, USA.
RNA. 2023 Dec;29(12):1960-1972. doi: 10.1261/rna.079825.123. Epub 2023 Oct 4.
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.
无细胞蛋白质合成 (CFPS) 系统可方便地在体外表达具有许多科学、工业和治疗应用的蛋白质。在这里，我们展示了一种经过优化的、高效的人类无细胞翻译系统，该系统绕过了目前使用的体外系统的许多限制。该 CFPS 系统基于内源性表达 GADD34 和 K3L 蛋白的人 HEK293T 细胞提取物，这两种蛋白可抑制翻译起始因子 eIF2α 的磷酸化。在细胞裂解物制备之前在人细胞中过表达 GADD34 和 K3L 辅助蛋白可显著简化裂解物制备。我们发现，在细胞裂解之前表达 GADD34 和 K3L 辅助蛋白可使提取物中 eIF2α 的磷酸化水平保持在较低水平。在体外翻译反应中，eIF2α 的磷酸化以 GCN2 依赖性的方式适度增加，GCN2 激酶抑制剂可抑制这种磷酸化。这个新的 CFPS 系统应该有助于在细胞外更接近生理条件下探索人类翻译机制。
