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.
Highlights
Cell-free protein synthesis (CFPS) is an emerging research field
We describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells
We shared our detailed protocols for the design, development and optimization of a functional mammalian cell-free protein synthesis platform derived from cultured Chinese Hamster Ovary (CHO) cells
Summary
Cell-free protein synthesis (CFPS) is an emerging research field. Originally developed to decipher the genetic code (Nirenberg and Matthaei, 1961), CFPS has recently become a powerful tool, providing new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more (Gregorio et al, 2019). Mammalian systems are of particular interest due to their ability to produce glycoproteins with human-like N-linked glycosylation This has led to the development of CFPS platforms for human (Mikami et al, 2008; Yadavalli and Sam-Yellowe, 2015; Burgenson et al, 2018) and Chinese Hamster Ovary (CHO) cells (Brödel et al, 2014, 2015; Martin et al, 2017; Thoring et al, 2017; Thoring and Kubick, 2018) among others. Since the product details of the commercial kits are not disclosed and reagents are provided as pre-mixed stocks, they have limited flexibility for customizing the reaction for different purposes
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