Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572225
Title: Factors affecting the specific productivity of Pichia pastoris
Author: Aw, Rochelle
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Please try the link below.
Access through Institution:
Abstract:
Pichia pastoris has become one of the most popular recombinant protein expression platforms, despite the lack of understanding into the fundamentals of protein expression. Whilst P. pastoris exhibits high volumetric productivity, it has a low specific productivity, which can be further reduced by protein-specific problems. This thesis employs several strategies commonly used to increase specific productivity, and assesses their impact on the productivity and cell biology of P. pastoris. Gene dosage has been reported to increase titre; therefore multiple copies of human serum albumin were integrated into P. pastoris to assess the correlation between recombinant protein productivity and copy number. Post-transformational vector amplification was used to generate clones containing up to five copies of HSA. However it was not possible to correlate copy number and yield as 15 L bioreactor cultures showed significant genetic instability. The mean final copy number was 2.6 ± 1.0. Further work was undertaken to evaluate possible ways to prevent instability, such as different selection methods, mutation of RAD51 and RAD52 which are both possible RecA homologs and whether the locus of vector integration plays a part. Integration into the rDNA locus resulted in increased stability with a five copy clone averaging 3.8 ± 1.6. Furthermore, no clones showed complete loss of the integrated vector as observed with integration into the AOX1 locus. Additionally, the little understood phenomenon of clonal variation was investigated which has been reported to affect specific productivity. Nine clones, with a range of productivity, were chosen for transcriptomic analysis. Variation between different clones was not uniform, even within the high, mid and low secretor groups. However, the ER associated degradation pathway was consistently upregulated in the high secretors which could be exploited in the future for strain development and selection.
Supervisor: Leak, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.572225  DOI: Not available
Share: