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

作     者：Alex J.Spice Rochelle Aw Daniel G.Bracewell Karen M.Polizzi 

作者机构：Department of Chemical EngineeringImperial College LondonLondonUK Imperial College Centre for Synthetic BiologyImperial College LondonUK Department of Biochemical EngineeringUniversity College LondonLondonUK 

出 版 物：《Synthetic and Systems Biotechnology》 (合成和系统生物技术（英文）)

年 卷 期：2020年第5卷第3期

页      面：137-144页

核心收录：

学科分类：0710[理学-生物学] 0831[工学-生物医学工程（可授工学、理学、医学学位）] 1001[医学-基础医学(可授医学、理学学位)] 08[工学] 0703[理学-化学] 0836[工学-生物工程] 

基　　金：This research is funded by the Department of Health and Social Care using UK Aid funding and is managed by the Engineering and Physical Sciences Research Council(EPSRC grant number:EP/R013764/1). 

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

摘      要：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.