RT Journal Article
SR Electronic
T1 A highly efficient human cell-free translation system
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.02.09.527910
DO 10.1101/2023.02.09.527910
A1 Nikolay A. Aleksashin
A1 Stacey Tsai-Lan Chang
A1 Jamie H. D. Cate
YR 2023
UL http://biorxiv.org/content/early/2023/05/23/2023.02.09.527910.abstract
AB 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 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 in vitro 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.Competing Interest StatementThe authors have declared no competing interest.