Design, development and optimisation of a functional mammalian cell-free protein synthesis platform
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Published version
Author(s)
Type
Journal Article
Abstract
In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.
Date Issued
2021-02-02
Date Acceptance
2020-12-29
Citation
Frontiers in Bioengineering and Biotechnology, 2021, 8
ISSN
2296-4185
Publisher
Frontiers Media
Journal / Book Title
Frontiers in Bioengineering and Biotechnology
Volume
8
Copyright Statement
© 2021 Heide, Buldum, Moya-Ramirez, Ces, Kontoravdi and Polizzi.This is an open-access article distributed under the terms of the Creative CommonsAttribution License (CC BY). The use, distribution or reproduction in other forumsis permitted, provided the original author(s) and the copyright owner(s) are creditedand that the original publication in this journal is cited, in accordance with acceptedacademic practice. No use, distribution or reproduction ispermitted which does not comply with these terms.
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Sponsor
Engineering & Physical Science Research Council (EPSRC)
Engineering & Physical Science Research Council (E
Grant Number
EP/K038648/1
EP/R511547/1
Subjects
Chinese hamster ovary cells
cell extract
cell-free protein synthesis
coupled batch reactions
in vitro transcription-translation
synthetic biology
0699 Other Biological Sciences
0903 Biomedical Engineering
1004 Medical Biotechnology
Publication Status
Published
Article Number
ARTN 604091