A highly efficient human cell-free translation system.

Autor:	Aleksashin NA; 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., Chang ST; 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., Cate JHD; Innovative Genomics Institute, University of California-Berkeley, Berkeley, California 94720, USA j-h-doudna-cate@berkeley.edu.; Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, California 94720, USA.; Department of Chemistry, University of California-Berkeley, Berkeley, California 94720, USA.
Jazyk:	angličtina
Zdroj:	RNA (New York, N.Y.) [RNA] 2023 Dec; Vol. 29 (12), pp. 1960-1972. Date of Electronic Publication: 2023 Oct 04.
DOI:	10.1261/rna.079825.123
Abstrakt:	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. (© 2023 Aleksashin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
Databáze:	MEDLINE
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