Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

TL;DR

This paper introduces new methods to improve the production of difficult proteins using modified CHO cells and cell-free systems.

Contribution

The study introduces a cell-free system with T7 RNA polymerase and mutant eIF2α to enhance protein production without exogenous additives.

Findings

Modified CHO cell lysates with T7 RNA polymerase and mutant eIF2α increased protein yields in cell-free synthesis.

The Rosa26 site was identified as a potential location for integrating T7 RNA polymerase for cell-based expression.

The approach allows flexible switching between cell-free and cell-based protein production methods.

Abstract

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.

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