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

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.

A highly efficient human cell-free translation system

“…One of the most significant advantages of the translational system described here is that all components of the translational machinery are derived from human sources. While previous results in Chinese hamster ovary (CHO) cells that transiently overexpressed GADD34Δ also showed increases in the translational activity in a CFPS system ( 48 ), the use of CHO cells may introduce confounding variables to the study of aspects of human translation that are not conserved. Stable integration of the recombinant genes for GADD34Δ and K3L in the genome as described here should also enable more controlled preparation of cell extracts without complications due to transient transfection on cell viability ( 48 ).…”

“…While previous results in Chinese hamster ovary (CHO) cells that transiently overexpressed GADD34Δ also showed increases in the translational activity in a CFPS system ( 48 ), the use of CHO cells may introduce confounding variables to the study of aspects of human translation that are not conserved. Stable integration of the recombinant genes for GADD34Δ and K3L in the genome as described here should also enable more controlled preparation of cell extracts without complications due to transient transfection on cell viability ( 48 ). In the case of human derived CFPS systems, we find that HeLa translation systems can achieve high levels of translation (Fig.…”

Cell‐free protein synthesis system: A new frontier for sustainable biotechnology‐based products

“…141,142 Utilizing mammalian CFPS systems would be advantageous from a therapeutic standpoint because of the extract's lower immunogenicity, even though improvements in the complexity of products from prokaryotic systems are undoubtedly important. Despite some improvements in the creation of internal mammalian extracts, 143,144 commercial products are still quite expensive and are only offered as separate transcription or translation kits at the moment. In order to make standardized practices and goods more accessible, further work is still required.…”

Microbial protein cell factories fight back?

“…CFPS systems have been around for decades and are being improved and exploited as attractive alternatives to cell-based expression. Numerous CFPS crude cell extracts are being developed and/or used, ranging from prokaryotic [126,127] to eukaryotic [128,129] systems.…”