Chinese Hamster Ovary (CHO) cells are a desirable platform to produce the next generation of recombinant therapeutic proteins due to their ability to produce human-like post-translation modification. However, the expression of such proteins can stress CHO cells, leading to a limitation in productivity. Many strategies have been evaluated with the aim of increasing the protein production, where productivity around 10g/L has been achieved, but further improvement is required. This Thesis has focused on the identification of the molecular events responsible for impaired production via the utilisation of stress-inducing recombinant proteins under inducible conditions. The utilisation of the inducible system allowed the separation of cellular growth and protein production, which permits the direct evaluation of molecular changes related to the protein production. Evaluation of the T-REx system was performed and it was found that after tet addition protein production was induced, whilst no protein was observed in the absence of inducer. Generation of 3 different constructs was performed, containing: an easy-to-express protein, like Growth Hormone (GH); a difficult to express protein, as anti-trypsin Z (ATZ); and a protein that can be classified as difficult to express but is partially secreted such as anti-thrombin III (ATIII). These proteins were selected in order to observe the differences in cellular behaviour under different protein patterns. Generation of stable T-REx, containing inducible plasmid with each gene of interest, was performed. Evaluation of tet addition at different times was performed and it was observed that the addition of tet does not affect the cellular growth in the concentration used. Only the ATZ T-REx CHO cell line showed a change in cellular growth after tet addition, which suggests that cellular changes are related to the induction of protein production. Metabolic analysis was performed and no changes related to tet addition or protein production was found in the ATZ T-REx CHO cell line. Additionally, RNA-Seq analysis was performed at early stages (8h and 24h after tet addition) was performed. It was observed that the addition of tet did not affect the mRNA level, suggesting that the concentration used was enough to trigger protein production without having a cellular effect. Induction of ATZ showed an up-regulation of genes related to ER stress and ERAD, and suggests a possible up-regulation of autophagy. This data show us that the separation of cellular growth and protein synthesis can be a powerful tool towards the finding of new target(s) for the CHO cell engineering.
Date of Award | 1 Aug 2018 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Alan Dickson (Supervisor) & Anil Day (Supervisor) |
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- RNA-Seq
- ATZ
- CHO cells
- Inducible system
Measurement of protein burden in CHO cells by development of an inducible expression platform
Mosqueira Dinamarca, M. (Author). 1 Aug 2018
Student thesis: Phd