2021
DOI: 10.1021/acssynbio.1c00195
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Characterizing and Improving Reaction Times for E. coli-Based Cell-Free Protein Synthesis

Abstract: Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in highyielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across inhouse E. coli extracts and commercial kits. We then set out to finetune our in-house extract … Show more

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Cited by 13 publications
(12 citation statements)
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“…In general, the removal of lacO alone had a more substantial impact on sfGFP fluorescence and expression in the extract-based CFAI and NEB Express systems whereas, in the NEB PURExpress CFPS system, the removal of the N-terminal tags had the greatest effect. Consistent with prior observations, fluorescence data also revealed much higher maximum yields of sfGFP through the CFAI-based CFPS system (>1,000 μg/ml) than in the NEB Express (∼800 μg/ml) and NEB PURExpress (∼200 μg/ml) CFPS systems ( Burrington et al, 2021b ).…”
Section: Resultssupporting
confidence: 88%
“…In general, the removal of lacO alone had a more substantial impact on sfGFP fluorescence and expression in the extract-based CFAI and NEB Express systems whereas, in the NEB PURExpress CFPS system, the removal of the N-terminal tags had the greatest effect. Consistent with prior observations, fluorescence data also revealed much higher maximum yields of sfGFP through the CFAI-based CFPS system (>1,000 μg/ml) than in the NEB Express (∼800 μg/ml) and NEB PURExpress (∼200 μg/ml) CFPS systems ( Burrington et al, 2021b ).…”
Section: Resultssupporting
confidence: 88%
“…However, the presence of high concentrations of acetate or glutamate anions in the wash buffer during cell harvesting does impact the underlying metabolome of resulting cell extracts and could alter the proteome and observed gene expression activity through regulatory changes that occur prior to cell lysis, particularly during the runoff incubation. , Concentrations of CFE reagents and their byproducts must also be considered and optimized for different applications . For example, including polyamines (such as putrescine) and crowding reagents (such as polyethylene glycol and Ficoll) can improve CFE through nucleic interactions and/or molecular crowding effects, but these components can reduce protein synthesis above an optimal concentration. ,, In addition to these insights, the metabolomics data enable inferences into related compounds that could not be detected on this specific analytic pipeline, such as some phosphorylated metabolites.…”
Section: Resultsmentioning
confidence: 99%
“…62 For example, including polyamines (such as putrescine) and crowding reagents (such as polyethylene glycol and Ficoll) can improve CFE through nucleic interactions and/or molecular crowding effects, but these components can reduce protein synthesis above an optimal concentration. 54,66,69 In addition to these insights, the metabolomics data enable inferences into related compounds that could not be detected on this specific analytic pipeline, such as some phosphorylated metabolites.…”
Section: ■ Results and Discussionmentioning
confidence: 99%
“…Furthermore, since EF‐P binds to the 9 nt D‐loop found in tRNAPro isoacceptors, and not to proline itself (Katoh et al ., 2016), the use of this factor can be expected to play a key role in the precision of cell‐free translation engineering, a feature essential for SynBio developments. After half a century of hard work, the various obstacles to the development of in vitro synthesis continue to be improved, with the emphasis on speed (Burrington et al ., 2021). Much remains to be understood to allow the industrial process of translation to be stable in time, with proper maintenance activities in particular.…”
Section: In Vitro Syntheses and Evolutionmentioning
confidence: 99%