Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems.

ABSTRACT: Cell-free translation from human cells is a powerful platform for studying mammalian gene expression and building synthetic biology tools, but productivity is often curtailed by inhibitory phosphorylation of eIF2α on residue Ser52. Here we systematically explored complementary strategies to bypass this initiation block across editable and hard-to-edit human cell types. In Expi293F suspension cells, precise genome editing of EIF2S1 to block Ser52 phosphorylation (eIF2α-S52A) produced high-activity extracts. Genetic knockout of EIF2AK2 (PKR)-the principal eIF2α kinase engaged in eIF2α phosphorylation in Expi293F lysates-also improved translation, further establishing eIF2α phosphorylation as the dominant bottleneck in Expi293F translation extracts. Because genome editing is imp

SUBMITTER: Aleksashin NA 

PROVIDER: S-EPMC12767633 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature