Cell-free protein synthesis using Chinese hamster ovary cells.

Cell-free protein synthesis (CFPS) platforms can be used for rapid and flexible expression of proteins. The use of CFPS platforms from mammalian, specifically Chinese hamster ovary (CHO) cells, offers the possibility of a rapid prototyping platform for recombinant protein production with the capabilities of post-translational modifications. In this chapter, we discuss a refined CFPS system based on CHO cells, including: extract preparation, reaction mix composition, and accessory protein supplementation to enhance expression. Specifically, when the CHO cell extract is combined with a truncated version of GADD34 and K3L, stress-induced eIF2 phosphorylation is reduced and inhibition of translation initiation is relieved, increasing yields. A brief summary of the protocol for running the CFPS reactions is also described. Overall, the method is reliable and leads to a highly reproducible expression system. Finally, the advantages and disadvantages of the platform, in addition to expected outcomes, are also discussed.

Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform.

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.