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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

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Synthetic and Systems Biotechnology 2020年第3期5卷 137-144页

作者： Alex J.Spice Rochelle Aw daniel g.bracewell Karen M.Polizzi Department of Chemical Engineering Imperial College LondonLondonUK Imperial College Centre for Synthetic Biology Imperial College LondonUK Department of Biochemical Engineering University College LondonLondonUK

A renaissance in cell-free protein synthesis(CFPS)is underway,enabled by the acceleration and adoption of synthetic biology *** has emerged as a powerful platform technology for synthetic gene network design,biosensing and on-demand *** 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 ***,cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields,despite the potential for improved protein folding and posttranslational *** we describe further development of a Pichia pastoris cell-free platform,a widely used expression host in both academia and the biopharmaceutical *** 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 *** was achieved in a minimal number of experimental runs,within the constraints of the design and without the need for liquid-handling *** 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,*** study highlights the ***-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

关键词： Cell-free protein synthesis Pichia pastoris Synthetic biology Design of experiments(DOE)

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