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Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort

Spice, AJ; Aw, R; Bracewell, DG; Polizzi, KM; (2020) Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort. Synthetic and Systems Biotechnology , 5 (3) pp. 137-144. 10.1016/j.synbio.2020.06.003. Green open access

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Abstract

A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

Type: Article
Title: Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort
Location: China
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.synbio.2020.06.003
Publisher version: https://doi.org/10.1016/j.synbio.2020.06.003
Language: English
Additional information: Copyright © 2020 The Author(s). Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Keywords: Cell-free protein synthesis, Pichia pastoris, Synthetic biology, Design of experiments (DOE)
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10105549
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