Cell-free protein synthesis can circumvent the challenge of producing membrane proteins and toxic proteins in living cells. Its open environment permits the addition of specific components to enhance protein solubility and introduce novel chemical reactants, such as non-canonical amino acids, for site-specific protein modifications. Eukaryotic cell-free systems are particularly advantageous for producing complex proteins, as they support post-translational modifications and optimal protein folding conditions. However, cultivating mammalian cells, like the commonly used Chinese hamster ovary (CHO) cells for pharmaceutical protein production, is costly. This doctoral thesis aims to develop a cost-effective cell-free system for producing difficult-to-express proteins. To achieve this, an optimal gene location for stable CHO cell line development and subsequent preparation of translationally active cell lysate was identified. The efficiency of cell-free transcription was enhanced by incorporating T7 RNA polymerase into CHO cells, while translation initiation was improved by using CHO cells expressing a mutant of the α subunit of eukaryotic initiation factor 2. Additionally, straightforward orthogonal cell-free translation was achieved by introducing orthogonal aminoacyl tRNA synthetases into CHO cells, facilitating the cell-free production of site-specifically modified membrane proteins. Furthermore, the identified locus in the CHO genome was utilized to create a tetracycline-inducible expression system, enabling both cell-free and inducible cell-based production of challenging proteins at desired time points and cell densities. Another goal of this research was to develop a yeast-based cell-free system using Pichia pastoris. This system combines the benefits of low-cost production, a eukaryotic environment, and the opportunity to synthesize difficult-to-express proteins. Moreover, it could be demonstrated that statistical Design of Experiments can be performed in a short time and on a small scale to identify optimal reaction parameters, facilitating the rapid adjustment of required protein synthesis conditions. In summary, the established methods streamlined induced cell-based expression and cell-free protein synthesis by minimizing the need for external supplements, improving protein production, and reducing costs.
