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A highly efficient human cell-free translation system - PubMed

A highly efficient human cell-free translation system

Nikolay A Aleksashin et al. RNA. 2023 Dec.

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 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.

Keywords: cell-free translation system; protein synthesis; ribosome.

© 2023 Aleksashin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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Figures

FIGURE 1.
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 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.

FIGURE 2.
FIGURE 2.

Endogenously expressed GADD34Δ and K3L increase 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 nLuc 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.
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 the HEK293T-based CFPS systems with and without GADD34Δ and K3L expression. The uncapped EMCV IRES-containing polyadenylated mRNA was used to drive the synthesis of nLuc. (B) Induction of eIF2α phosphorylation in the HeLa-based commercial translation system supplemented with the same mRNA. For both A and B, the blue arrow indicates the nonphosphorylated form of the eIF2α, while the magenta arrow indicates the phosphorylated form on the Phos-tag gels. The western blots above the Phos-tag gels used normal SDS–PAGE gels sequentially blotted with anti-eIF2α and anti-P-Ser51 eIF2α antibodies. For both A and B, the gels are representative of two independent experiments. The percentage of eIF2α phosphorylation, based on the Phos-tag gels, is indicated under the gels (see Materials and Methods). (C) Western blot showing the amount of the eIF2B-ε and eIF2α in the HEK293T and HeLa cellular extracts. The gel is representative of two independent experiments. (D) The GCN2 but not PKR or PERK kinase inhibitor protects eIF2α from phosphorylation during CFPS. The compound A-92 (Brazeau and Rosse 2014) was used as a GCN2 kinase inhibitor, C-16 (Jammi et al. 2003) for PKR kinase inhibition, and GSK2606414 (Axten et al. 2012) as an inhibitor of PERK kinase. The concentrations of the eIF2α-specific kinase inhibitors are indicated. Gels are representative of two independent experiments. (E) Cell-free synthesis of nLuc in different concentrations of the eIF2α-specific kinase inhibitors. All error bars represent one standard deviation of three independent replicates.

FIGURE 4.
FIGURE 4.

Identification of possible additional 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 HBB and uncapped EMCV IRES-containing polyadenylated mRNAs were used to drive the synthesis of nLuc. The gels are representative of two independent experiments.

Nikolay Aleksashin
Nikolay Aleksashin

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