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Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions

Lookup NU author(s): Dr Alice BanksORCiD, Dr Colette Whitfield, Dr Steven Brown, Dr David Fulton, Dr Dennis LendremORCiD, Dr Thomas HowardORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


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.

Publication metadata

Author(s): Banks AM, Whitfield CJ, Brown SR, Fulton DA, Goodchild SA, Grant C, Love J, Lendrem D, Fieldsend JE, Howard TP

Publication type: Article

Publication status: Published

Journal: Computational and Structural Biotechnology Journal

Year: 2022

Volume: 20

Pages: 218-229

Online publication date: 13/12/2021

Acceptance date: 08/12/2021

Date deposited: 16/12/2021

ISSN (electronic): 2001-0370

Publisher: Elsevier


DOI: 10.1016/j.csbj.2021.12.013


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