Investigating the Role of TOR Signalling, and the Link between Actin Binding Protein Cofilin and Mitochondrial Function and their Effects on Recombinant Protein Production

Due to the increasing demand in Biotherapeutics, this thesis focussed on investigating novel ways to exploit major signalling pathways to enhance recombinant protein production (rP) in yeast and CHO cell expression systems. The expression of model recombinant proteins in yeast allowed us to screen for specific targets located within important environmental signalling pathways for effects upon recombinant protein production. Targets identified within the yeast system and that were conserved were then tested within CHO cell expression system. One of the systems investigated with regards to rP production was target of rapamycin (TOR) pathway. TOR signalling is a global regulator of nutrient sensing and energy status. It is a highly conserved Ser/Thr protein kinase pathway which is involved in processes such as protein synthesis, ribosome biogenesis, cell cycle, gene transcription, autophagy, and metabolism. As TOR is heavily involved in the control of protein synthesis, we asked whether it could be manipulated to enhance recombinant protein production (rP). We identified that TOR mediated amino acid sensing and autophagy are important for maintaining rP production in both yeast and CHO cells systems. Previous work supporting this thesis suggests that the actin binding protein cofilin can be manipulated to enhance both mitochondrial function and environmental sensing. We hypothesised that Cofilin functions may be useful in maintaining mitochondrial function during the stress imposed by rP production. Using a library of Cofilin strains expressing mutant alleles, we found that the control of MAPK signalling, and in particular the downstream effector Ste12, and mitochondrial functionality have a significant effect on GLuc expression when driven by the mating factor signal sequence. Manipulation of mitochondrial function or STE12 function also led to changes in rP production. In many case changes in Gluc expression or secretion were not replicated in a separate Killer toxin secretion system. As we did not see the same effects in alterations to killer toxin secretion, we concluded that these findings were specific to the presence of the signal sequence from the mating alpha factor. The manipulation of MAPK signalling and mating factor signal sequence driven secretion would appear to be a promising avenue for further enhancement of the yeast system. This work highlights the complex nature of manipulating signalling networks to achieve improvements in rP yield. However a better understanding of the mechanisms involved and the ability to manipulate these changes/responses will be beneficial for enhanced protein production from yeast and mammalian expression systems.