Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.
Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.
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DOI：
10.1016/j.csbj.2021.12.013
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发表时间：
2022
影响因子：
6
通讯作者：
Howard TP
中科院分区：
文献类型：
--
作者：
Banks AM;Whitfield CJ;Brown SR;Fulton DA;Goodchild SA;Grant C;Love J;Lendrem DW;Fieldsend JE;Howard TP
新型无细胞蛋白质合成反应缓冲液将性能提高了400%。在不同蛋白质的合成中保持增强的性能。不同细胞裂解物批次和E.大肠杆菌菌株。缓冲液组分以不同的方式影响反应动力学的各个方面。无细胞蛋白质合成（CFPS）反应已经越来越受欢迎，特别是在基因构建原型，生物传感器技术和新化学蛋白质的生产等应用中。工作经常集中在优化CFPS协议，以提高蛋白质产量，降低成本，或开发精简的生产协议。在这里，我们描述了一个流行的CFPS反应缓冲液的20个组件的统计实验设计分析。我们同时确定影响蛋白质产量，反应速率，滞后时间和反应寿命的因素和因素相互作用。这种系统性的实验方法能够创建一个统计模型，捕捉CFPS反应的多种行为，以响应组分及其相互作用。我们表明，一种新型的反应缓冲液比参考反应的性能高出400%，并且在细胞裂解物、大肠杆菌菌株和其他细胞裂解物的批次中显著减少了CFPS的失败。大肠杆菌，以及不同蛋白质的合成。对反应组分如何影响动力学响应和鲁棒性的详细和定量理解对于未来部署无细胞技术至关重要。
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.
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影响因子：
3.7
作者：
Ge X;Luo D;Xu J
通讯作者：
Xu J
影响因子：
4.6
作者：
Kwon YC;Jewett MC
通讯作者：
Jewett MC
影响因子：
3.7
作者：
Kasli, Ikhlaas M.;Thomas, Owen R. T.;Overton, Tim W.
通讯作者：
Overton, Tim W.
影响因子：
5.2
作者：
Kay, Jennifer E.;Jewett, Michael C.
通讯作者：
Jewett, Michael C.
DOI：
10.1023/b:jsfg.0000029204.57846.7d
发表时间：
2004-01-01
期刊：
Journal of Structural and Functional Genomics
影响因子：
--
作者：
Kigawa, Takanori;Yabuki, Takashi;Yokoyama, Shigeyuki
通讯作者：
Yokoyama, Shigeyuki