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Reprogramming of Sterol Biosynthesis in Chinese Hamster Ovary Cells for Enhanced Recombinant Protein Production

AJAYI, FOLASADE ABIOSE (2019) Reprogramming of Sterol Biosynthesis in Chinese Hamster Ovary Cells for Enhanced Recombinant Protein Production. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:79941)

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Abstract

The biopharmaceutical industry is a multi-billion dollar global market with projections that within the next 5 to 10 years, up to 50% of all drugs in development will be biopharmaceuticals. The majority of these drugs are recombinantly produced protein based therapeutics with the demand for such new therapeutic products continuing to increase, broadening the range of medical conditions that they are being used to treat. Chinese hamster ovary (CHO) cells are the preferred and most commonly

perceived cellular stress as a result of the load imposed on the cell by the recombinant protein.

nuclear envelope (NE) expansion when over-expressed in mammalian cell lines. The work described here therefore set out to determine whether overexpression of these sterol reductases could enhance the secretory capacity of CHO cells by expansion of the ER and hence lead to increased yields of difficult to express recombinant proteins compared to those cells where these were not overexpressed. The TM7SF2 and DHCR7 genes were cloned into a mammalian expression vector with a hygromycin selection marker to allow for the selection of stable cell lines where the genes had been integrated into the genome. Stable CHO cells overexpressing TM7SF2 and DHCR7 were generated to

expression levels of the sterol reductases were determined in heterogeneous pools and then used for limited dilution cloning to isolate clonal cell lines with differing sterol reductase expression which were further investigated. Growth profile assays were undertaken to monitor any toxicity effect of over-expression of TM7SF2 and DHCR7 on CHO cells. Transient expression studies of the recombinant bio therapeutic protein etanercept, a 150 kDa biomolecule and erythropoietin (EPO), a 36 kDa recombinant protein, were carried out to investigate the production capacity of lipid engineered CHO cells with reference to controls. Immunocytolocalisation studies were also undertaken to investigate pools and clones of lipid targets predicted to be ER-localized or associated. A YFP tag was present on the N-terminal of TM7SF2 and DHCR7 molecules and was used to monitor expression by fluorescence in stable CHO cells.

of secreted recombinant target protein in the cell culture supernatant. Confocal images also showed clones expressed different levels of YFP signal which related to expression levels observed when western blot techniques were used. In summary, the data presented here shows that the manipulation of cellular circuits such as cholesterol biosynthesis has the potential to enhance cellular growth and recombinant protein yield. By designing new hosts and cellular circuits to reprogramme

the CHO cell ER there is the potential of expanding the secretory capacity and/or subsequent secretory vesicle system.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Smales, Mark
Uncontrolled keywords: Transmembrane 7 Super family member 2, 7-dehydrocholesterol reductase, Cholesterol Biosynthesis, Recombinant Proteins, Chinese Hamster Ovary cells.
Divisions: Faculties > Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 04 Feb 2020 11:10 UTC
Last Modified: 05 Feb 2020 09:29 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/79941 (The current URI for this page, for reference purposes)
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