Elimination and characterization of a Tetrahymena thermophila protease gene that can reduce recombinant drug production efficiency by gene knock-out method

In this thesis, the hypothesis that naturally secreted proteases of T. thermophila degrede protein-structured recombinant biopharmaceuticals secreted externally, and that reduce production efficiency has been tested. Five of highly expressed secretome protease genes were determined via literature research and bioinformatics approaches, and CTH34 gene was deleted by somatic homologous recombination while CYP6, CTH40, CTH90 and CTH115 were deleted by co-deletion, followed by mutant strain confirmation using PCR after antibiotic selection. The proteolytic activity of wild type and mutant culture secretomes was measured in vitro assays against casein and rituximab substrates, and substrate degradation was confirmed by SDS-PAGE. While secretomal proteolytic activity decreased by 34.3% in the CTH34-KO strain, cell number of the flask population in the logarithmic and stationary phase of the growth curve also decreased by ~50%. Besides, it is observed that degradation of the rituximab substrate was significantly reduced by CYP6-KO strain secretome compared to wild type whereas the cell number in the stationary phase of the growth curve also diminished by ~25%. According to forementioned findings, it was concluded that T. thermophila can be efficiently tailored for recombinant protein drug production by deletion of protease genes such as CTH34 and CYP6 from the genome.