Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.790129
Title: An upstream platform for the production of high grade heterologous proteins in the yeast Pichia pastoris
Author: Woodhouse, S. A.
ISNI:       0000 0004 8503 4559
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
Pichia pastoris is a methylotrophic yeast and well established expression system for the production of therapeutic proteins and industrial enzymes. It is characterised by its tightly regulated promoters, natural ability to secrete recombinant proteins and potential to grow to very high cell densities in order to combat low product titres. These high cell densities, in combination with the requirement of methanol as an inducing agent, lead to problems, however, with cell viability being negatively influenced, resulting in the release of host cell proteins, which include proteases that both reduce product quality and complicate purification. This thesis introduces a simple fermentation strategy that reduces the amount of methanol required during fermentation by utilising the non-repressing substrate sorbitol as a co-feed with methanol during induction. This allowed cell growth profiles to remain similar to those seen with current protocols, with final cell densities of 120-140 g/L dry cell weight (DCW) being attained. Cell viability and product yields were unaffected by the new strategy, but protease release was reduced due to a shift in host cell protein impurity profiles. The scalability of fermentations was also greatly increased due to a 60% reduction in heat generation during induction. Finally, a methodology for the determination of cellular robustness was successfully developed using adaptive focussed acoustics: this was used to demonstrate that methanol induction did not negatively impact cellular robustness, and that reduced growth rates were a key requirement of enhanced cellular robustness. These results have all been demonstrated with two strains of P. pastoris, an in-house GS115 strain expressing secreted embryonic alkaline phosphatase (SEAP) and an industrially utilised strain, CLD804 expressing aprotinin.
Supervisor: Keshavarz-Moore, E. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.790129  DOI: Not available
Share: