A highly efficient human cell-free translation system

Abstract

Cell-free protein synthesis (CFPS) systems enable easyin vitroexpression 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 usedin vitrosystems. 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 rather than adding them after the fact significantly simplifies 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. Duringin vitrotranslation 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 translation 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.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

“…In the present study, we demonstrated that cell-free protein synthesis based on CHO cell lysates can be more efficient and cost-effective by modifying the host cells. The potential of enhancing cell-free cap-dependent translation initiation was recently demonstrated by overexpression of GADD34 and K3L in human cells [ 48 ]. It was shown that increased levels of the truncated protein phosphatase GADD34 and the vaccinia virus protein K3L prior to cell disruption resulted in decreased eIF2α phosphorylation and increased cell-free protein production based on human cell lysate.…”

JUNmRNA Translation Regulation is Mediated by Multiple 5’ UTR and Start Codon Features

“…In vitro translation reactions were performed using HEK293T pSB-HygB-GADD34-K3L translation-competent cell extract, as previously described (Aleksashin et al 2023). Translation reactions contained 50% translation-competent cell extract, 52 mM HEPES pH 7.4 (Takara), 35 mM potassium glutamate (Sigma), 1.75 mM Mg(OAc)2 (Invitrogen), 0.55 mM spermidine (Sigma), 1.5% Glycerol (Fisher Scientific), 0.7 mM putrescine (Sigma), 5 mM DTT (Thermo Scientific), 1.25 mM ATP (Thermo Fisher Scientific), 0.12 mM GTP (Thermo Fisher Scientific), 10 mM L-Arg; 6.7 mM each of L-Gln, L-Ile, L-Leu, L-Lys, L-Thr, L-Val; 3.3 mM each of L-Ala, L-Asp, L-Asn, L-Glu, Gly, L-His, L-Phe, L-Pro, L-Ser, L-Tyr; 1.7 mM each of L-Cys, L-Met; 0.8 mM L-Trp, 20 mM creatine phosphate (Roche), 60 µg/mL creatine kinase (Roche), 4.65 µg/mL myokinase (Sigma), 0.48 µg/mL nucleoside-diphosphate kinase (Sigma), 0.3 U/mL inorganic pyrophosphatase (Thermo Fisher Scientific), 100 µg/mL total calf tRNA (Sigma), 0.8 U/μL RiboLock RNase inhibitor (Thermo Scientific), and 1000 ng of the corresponding mRNA.…”

“…HEK293T pSB-HygB-GADD34-K3L cells (Aleksashin et al 2023) were maintained in DMEM media (Gibco) supplemented with 10% Tet-system approved FBS (Gibco) and 1% Pen/Strep (Gibco). Cells were grown at 37 °C in 5% carbon dioxide and 100% humidity.…”

“…Cells were grown for extract preparation as follows. The day after plating cells In vitro translation extracts were made from HEK293T pSB-HygB-GADD34-K3L cells using a previously described protocol (Aleksashin et al 2023). Cells were placed on ice, scraped and collected by centrifugation at 1000 xg for 5 minutes at 4 °C.…”

“…In vitro translation reactions were performed using HEK293T pSB-HygB-GADD34-K3L translation-competent cell extract, as previously described (Aleksashin et al 2023).…”