Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions
| Autor: | Alice M. Banks, Colette J. Whitfield, Steven R. Brown, David A. Fulton, Sarah A. Goodchild, Christopher Grant, John Love, Dennis W. Lendrem, Jonathan E. Fieldsend, Thomas P. Howard |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
CoA
coenzyme A RFU relative fluorescence units ATP adenosine triphosphate Mg magnesium glutamate Biophysics CTP cytidine triphosphate Biochemistry CFE cell-free extract OFAT one-factor-at-a-time Statistical engineering Automation GTP guanosine triphosphate NAD nicotinamide adenine dinucleotide Structural Biology X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside Genetics tRNA transfer ribonucleic acid RSM Response Surface Model Robustness K-glutamate potassium glutamate 0802 Computation Theory and Mathematics ComputingMethodologies_COMPUTERGRAPHICS CFPS cell-free protein synthesis 0103 Numerical and Computational Mathematics LB lysogeny broth HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid PEP phosphoenolpyruvate Design of Experiments (DoE) PEG-8000 polyethylene glycol 8000 FEU fluorescein equivalent units Computer Science Applications cAMP cyclic adenosine monophosphate NTP nucleoside triphosphate eGFP enhanced green fluorescent protein DTT dithiothreitol G-6-P glucose-6-phosphate 3-PGA 3-phosphoglyceric acid DSD Definitive Screening Design Cell-free protein synthesis (CFPS) UTP uridine triphosphate DoE Design of Experiments TP248.13-248.65 Research Article Biotechnology |
| Zdroj: | Computational and Structural Biotechnology Journal, Vol 20, Iss, Pp 218-229 (2022) Computational and Structural Biotechnology Journal |
| ISSN: | 2001-0370 |
| Popis: | Graphical abstract Highlights • Novel cell-free protein synthesis reaction buffer improves performance by 400%. • Enhanced performance is maintained across the synthesis of different proteins. • Protein synthesis performance is robust across different cell lysate batches and E. coli strains. • Buffer components affect aspects of reaction kinetics in differing ways. Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies. |
| Databáze: | OpenAIRE |
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