Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

Abstract

Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared to the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces ~4-fold more total protein. However the majority of these polypeptides are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3 rd of that measured using the S30 crude extract system. Both of the two systems suffer from low ribosome recycling efficiency when translating proteins from 90 kD to 220 kD. A systematic fedbatch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination prior to energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while accumulation of inorganic phosphate contributes to reaction termination. Additionally, while adding EF-P to PURE reduced total protein translated, it also increased the fraction of active product and reduced partial translated product probably by slowing down the translation process.Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.peer-reviewed)

Anomalous Scaling of Gene Expression in Confined Cell-Free Reactions

“…Although such surface-induced repression of TXTL in a cell-sized droplet is observed in emulsified droplet, CFGE from the PURE system in liposomes 33 , 34 appears to show normal scaling behavior of I GFP ∝ R 3 . The PURE system has greater stability against mRNA degradation rather extract-based system 35 , 36 the approximation is reasonable as well. Such discrepancy may result from the difference of physical nature of CFGE expression systems: TXTL, used in this work, is based on an extract that is rather crowded due to the presence of macromolecules (10 mg/ml of proteins and 2% PEG8000).…”

Modelling cell-free RNA and protein synthesis with minimal systems

“…The strategies deployed here to increase the PURE system productivity could be combined with previous attempts. Owing to its modularity, the PURE system composition could be tuned by adjusting the concentration of various components, such as tRNAs, ribosomes, some initiation and elongation factors and magnesium acetate (74,92,93), or by replenishing low-molecular weight substrates (74,93,94). Other factors that are not present in the original composition have also been supplemented, e.g.…”

“…Other factors that are not present in the original composition have also been supplemented, e.g. the bovine serum albumin, crowding agents, EF-P (93,95,96), and proteins that recycle stalled ribosomes (93). Regarding the addition of ribosome rescuing proteins, the absence of detectable amounts of degraded mRNA by nuclease contaminants in PUREfrex suggests that implementation of rescuing systems, such as tmRNA and ArfA, will only have moderate influence.…”

A self-regenerating synthetic cell model

“…These estimates do not yet include ribosome or tRNA synthesis. Current approaches to optimizing transcription-translation systems mainly focus on increasing component concentrations or adding components to the system which can give rise to overall higher synthesis yields but consequently also require higher synthesis rates to achieve self-regeneration [24,25,31]. Instead, optimizing protein synthesis rates and the ratio of protein synthesis rate to total amount of protein contained in the system will be important for development of a biochemical universal constructor.…”

“…Moreover, it was recently shown that co-expression of multiple PURE components in a single batch reaction yielded the required concentrations for selfreplication [15]. However, these experiments didn't determine whether proteins were functionally synthesized, which varies largely for proteins expressed in the PURE system [30,31]. Two other studies showed that the 30S ribosomal subunit [22], and nineteen of twenty aaRSs, can be functionally synthesized in the PURE system [32].…”