Modelling as a tool for increasing the specific productivity of single-chain antibody fragments from Pichia pastoris
File(s)
Author(s)
Royle, Kate
Type
Thesis
Abstract
Pichia pastoris is a commonly used recombinant protein expression host, predominantly due to ease of genetic manipulation and its capacity for high cell densities in cheap culture media. While considerable yields can be achieved in this way, the specific productivity is relatively low. Consequently, the full impact of this host on industrial biotechnology has not yet been realised.
This research aimed to develop a strategy to optimise production of single chain antibody fragments (scFvs), an industrially relevant protein, using an integrated modelling and experimental approach. Initially, a dynamic model was constructed from literature sources to reproduce the scFv production pathway in P. pastoris. It incorporated aspects of transcription, translation, folding and misfolding in the endoplasmic reticulum (ER). Moreover, the unfolded protein response (UPR) and ER-associated degradation (ERAD) were added as these two stress pathways are crucial to productivity. Simulations qualitatively reproduced phenomena including secretion saturation and the negative influence of high gene copy numbers on yield.
The model was used to target evaluation of the experimental system: P. pastoris strains expressing the scFvs BC1 and MFE23. RT Q-PCR and LC-MS/MS results revealed some surprising correlations between certain factors, such as the concentration of Kar2 and PDI, and yield. Moreover, it showed that there was more than one route to high productivity. Finally, it suggested that there may be an internal regulation of Kar2 that would be crucial to strategies aiming to increase yield through overexpression of that chaperone.
Together, the results revealed a more complex picture of productivity than previously understood. In order to develop a strategy for optimal scFv production in P. pastoris, a greater understanding of the underlying biology and biochemistry is required. This research has suggested targets for future work which should generate insight into the network of factors responsible.
This research aimed to develop a strategy to optimise production of single chain antibody fragments (scFvs), an industrially relevant protein, using an integrated modelling and experimental approach. Initially, a dynamic model was constructed from literature sources to reproduce the scFv production pathway in P. pastoris. It incorporated aspects of transcription, translation, folding and misfolding in the endoplasmic reticulum (ER). Moreover, the unfolded protein response (UPR) and ER-associated degradation (ERAD) were added as these two stress pathways are crucial to productivity. Simulations qualitatively reproduced phenomena including secretion saturation and the negative influence of high gene copy numbers on yield.
The model was used to target evaluation of the experimental system: P. pastoris strains expressing the scFvs BC1 and MFE23. RT Q-PCR and LC-MS/MS results revealed some surprising correlations between certain factors, such as the concentration of Kar2 and PDI, and yield. Moreover, it showed that there was more than one route to high productivity. Finally, it suggested that there may be an internal regulation of Kar2 that would be crucial to strategies aiming to increase yield through overexpression of that chaperone.
Together, the results revealed a more complex picture of productivity than previously understood. In order to develop a strategy for optimal scFv production in P. pastoris, a greater understanding of the underlying biology and biochemistry is required. This research has suggested targets for future work which should generate insight into the network of factors responsible.
Version
Open Access
Date Issued
2013-09
Date Awarded
2014-05
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Leak, David
Kontoravdi, Cleo
Bundy, Jake
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)