A highly efficient human cell-free translation system

Cell-free protein synthesis (CFPS) systems enable easy 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 systems. This CFPS system i...

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Bibliographic Details
Published inbioRxiv : the preprint server for biology
Main Authors Aleksashin, Nikolay A, Chang, Stacey Tsai-Lan, Cate, Jamie H D
Format Journal Article
LanguageEnglish
Published United States 23.05.2023
Online AccessGet more information

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Summary:Cell-free protein synthesis (CFPS) systems enable easy 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 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 significantly simplifies cell lysate preparation. The new CFPS system improves the translation of 5' cap-dependent mRNAs as well as those that use internal ribosome entry site (IRES) mediated translation initiation. 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 translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. We also find evidence for activation of regulatory pathways related to eukaryotic elongation factor 2 (eEF2) phosphorylation and ribosome quality control in the extracts. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.