A highly efficient human cell-free translation system

Abstract

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.

Figure 1.. 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 N-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 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 (ECE) but not in the WT HEK293T cells, 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 GADD34Δ and K3L expressing (ECE) and WT HEK293T cell extracts programmed with an EMCV IRES-containing mRNA encoding nanoluciferase. All error bars represent one standard deviation of three independent replicates. (E) and (F) Representative polysome profiles of the WT HEK293T cell extract (E) and ECE (F) in the absence or presence of EMCV IRES containing nLuc mRNA template. G, 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.

Figure 2.. Endogenously expressed GADD34Δ and K3L increases translational activity comparable to the addition of exogenously expressed accessory proteins.

A, Western blot showing the amount of the GADD34Δ expressed in the engineered HEK293T cells and supplemented in the HeLa-based commercial translation system. The asterisk indicates a nonspecific band in the HeLa extract. The gel is representative of two independent experiments. B, A time course of nanoluciferase synthesis in the CFPS systems prepared based on the engineered HEK293T cell extract and HeLa-based extract with recombinant GADD34Δ and K3L supplement. All error bars represent one standard deviation of three independent replicates. C, Cell-free synthesis of GFP in the two translation systems. Orange bars represent the HeLa-based extract with exogenous GADD34Δ and K3L added, while the white bars represent the engineered HEK293T cell extract with endogenously expressed GADD34Δ and K3L proteins. All error bars represent one standard deviation of three independent replicates.

Figure 3.. GCN2 kinase is responsible for the residual phosphorylation of eIF2α during CFPS in the engineered HEK293T extract.

A, Western blots for phosphorylation of eIF2α in CFPS systems prepared based on the engineered and WT HEK293T cell extract. The capped human β-globin (HBB) and uncapped EMCV IRES-containing polyadenylated mRNAs were used to drive the synthesis of nanoluciferase. B, Induction of eIF2α phosphorylation in the HeLa-based commercial translation system supplemented with the same mRNAs. For both A and B, the blue arrow indicates the non-phosphorylated form of the eIF2α, while the magenta arrow indicates the phosphorylated form on the phos-tag gels. For both A and B, the gels are representative of two independent experiments. The percentage of eIF2α phosphorylation, based on the phos-tag gels, are indicated under gels (see Materials and Methods). C, The GCN2 but not PKR or PERK kinase inhibitor protects eIF2α from phosphorylation during CFPS. The compound A-92 (40) was used as a GCN2 kinase inhibitor, C-16 (41) for PKR kinase inhibition, and GSK2606414 (42) as an inhibitor of PERK kinase. The concentrations of the eIF2α-specific kinase inhibitors are indicated. Gels are representative of two independent experiments. D, Cell-free synthesis of nanoluciferase in different concentrations of the eIF2α-specific kinase inhibitors. All error bars represent one standard deviation of three independent replicates.

Figure 4.. Identification of possible CFPS-limiting factors.

A, Sucrose gradient sedimentation analysis reveals the presence of disomes and trisomes in the CFPS system based on the extract from the engineered HEK293T cell line, independent of exogenously added mRNA. B, Phosphorylation of eEF2 on T56 in the different translation systems. The capped human β-globin (HBB) and uncapped EMCV IRES-containing polyadenylated mRNAs were used to drive the synthesis of nanoluciferase. The gels are representative of two independent experiments. C, The proposed model for factors limiting cell-free protein synthesis in the engineered human cell extract. Phosphorylation of eIF2α inhibits translation initiation and can be bypassed by the overexpression of GADD34Δ and K3L proteins. Several factors may limit production of an even more active CFPS system by affecting translation elongation, including induction of the phosphorylation of eEF2 and activation of ribosome-associated quality control factors.