Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578056
Title: Analysis and exploitation of GPI anchors in the expression of recombinant protein biopharmaceuticals
Author: Riley, Aidan
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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Abstract:
In this thesis we show the generation and characteriation of specific GPI-anchored cell surface markers. The creation of these constructs has also allowed the quantification of levels of GPI linked protein shedding into the cell-culture media. Experiments presented in the thesis also show that the addition of a GPI anchor to Growth hormone (GH) appears to reduce expression in the media and that these molecules retain the GPI moiety. We speculated that anchoring cytokine ligands to the cell surface might also modulate receptor mediated signalling. To test the effect of GPI modification on cytokine ligands we fused the GH, and Ob mRNA sequences to the GPI anchor signal sequence of the naturally GPI-anchored protein, Thy-l. To our surprise we found that endogenously expressed GPI anchored cytokines acted as potent non-competitive cytokine receptor antagonists. Furthermore, we demonstrate that GPI anchored GH from a stable CHO cell-line can be purified and reinserted into mammalian cell membranes where they are able to induce receptor mediated signalling. Mammalian cell expression technologies are reliant on methods for obtaining high-yielding stable clones. This thesis describes the development of a high-throughput screening procedure based on the direct pull-down and enrichment of stably transfected cells using magnetic activared cell sorting (MACS). Results presented in this thesis demonstrate that it may be possible to use this procedure to isolate transfected cells from a heterogeneous population and to preferentially recover those rare cell populations which express higher levels of recombinant biopharmaceutical. Thus, GPI anchored markers may be used to facilitating facile cloning and identify population heterogeniety.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.578056  DOI: Not available
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