Fig 1: Blocking phosphorylation of eIF2α, but not eEF2, enhances translational efficiency in human Expi293F extracts.(A) Left: Phosphorylation of eIF2α on Ser52 converts eIF2 into an inhibitor of its guanine nucleotide exchange factor (eIF2B), blocking ternary complex regeneration and suppressing translation initiation. Right: Phosphorylation of eEF2 on Thr57 by eEF2 kinase (eEF2K) prevents ribosomal translocation, thereby slowing peptide elongation. (B) Phosphorylation of eIF2α (Ser52) by stress-activated kinases (PKR, GCN2, PERK, HRI) converts active eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, thereby preventing ternary complex formation and suppressing global initiation. (C) The eEF2 cycle: eEF2K phosphorylates eEF2 at Thr57, which prevents ribosomal translocation; dephosphorylation by PP2A restores elongation. (D) Illustration of the experimental workflow for lysate generation and in vitro translation (IVT). Suspension Expi293F cells were harvested, lysed under native conditions, and the resulting extracts programmed with a Nanoluciferase (NanoLuc) reporter mRNA to quantify translational efficiency. (E) Schematic representation of human Expi293F cells engineered by prime editing (PE) to introduce phospho-null substitutions in EIF2S1 (eIF2α S52A) and EEF2 (eEF2 T57A). All amino acid residue numbers correspond to the human reference sequences according to the UniProt database, entries P05198 and P13639 for EIF2S1 and EEF2, respectively. (F) Comparison of translational output in IVT reactions programmed with NanoLuc mRNA using extracts prepared from wild-type Expi293F cells or genome-edited Expi293F eIF2α-S52A and eEF2-T57A lines. All experiments were performed in biological triplicates.