Endogenously expressed GADD34Δ and K3L increase in vitro translation activity of the human cell extract. (A) Schematic of the role of GADD34 and K3L in counteracting eIF2α phosphorylation by eIF2α kinases. (B) Diagram of the sleeping beauty-based construct used for expression of GADD34Δ (which lacks the amino-terminal 240 amino acids) and K3L, which was integrated into the genome of HEK293T cells. TRE denotes a tetracycline (or doxycycline) responsive promoter that controls the expression of GADD34Δ and K3L, separated by the P2A sequence. The synthetic constitutive promoter RPBSA drives the expression of the fusion construct of a tet repressor together with the hygromycin resistance gene, separated by the P2A sequence. (C) Western blot showing expression of GADD34Δ in the engineered cell extract upon doxycycline-dependent induction, with ribosomal protein eS19 serving as a loading control. The gel is representative of two independent experiments. (D) A time course of nanoluciferase (nLuc) synthesis in the CFPS systems prepared based on the extracts with or without GADD34Δ and K3L expression. All error bars represent one standard deviation of three independent replicates. On the bottom, the western blot shows the absolute amount of synthetized nLuc in each of the translation systems with ribosomal protein eS19 serving as a loading control. The gel is representative of two independent experiments. (E) Representative polysome profiles of the CFPS based on the extracts with GADD34Δ and K3L expression in the absence or presence of EMCV IRES-containing nLuc mRNA template. (F) Nanoluciferase levels from cell-free translation reactions including polyadenylated nLuc mRNAs containing different 5′ UTRs, as indicated. All templates were uncapped, except the human β-globin (HBB) 5′ UTR.
