Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439301
Title: Physicochemical characterisation of disease associated abnormal prion proteins
Author: Stone, Lisa Ann
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2006
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Abstract:
The central feature of prion disease is the conversion of normal host-encoded cellular prion protein, PrPc, to an abnormal isoform, PrP. PrP80 appears to be the principal, if not the sole, component of infectious prions. Prion strain diversity seems to be encoded within PrP itself through a combination of PrP conformation, glycosylation and possibly other, as yet, unidentified post-translational modifications. Efficient purification of PrP will facilitate detailed chemical characterisation and investigation of conformations or assembly states required for prion infectivity. Purification of denatured PrP has been achieved through enrichment from brain homogenate using selective precipitation followed by chemical and thermal denaturation and isolation by immunoafTinity chromatography. These methods have isolated PrP from rodent prion strains with a yield of 50 % and purity of > 90 % of total protein and for the first time permit isolation of full length denatured PrPSc facilitating comprehensive structural analysis. Purified denatured PrP is expected to have all the covalent post-translational modifications that may be required for prion infectivity. In attempts to reconstitute prion infectivity, denatured PrP was refolded under different solvent conditions and assessed for changes in solubility, resistance to proteolytic digestion and infectivity in bioassay. Although distinct changes in the physicochemical properties of PrP were observed, to-date no evidence for reconstitution of prion infectivity has been found. In the course of these studies, it was discovered that Cu2f ions inhibit proteinase K activity and a detailed kinetic description of this inhibition was obtained. The lack of prion infectivity seen in refolded preparations of denatured PrP suggests a requirement for other as yet unidentified co-factors. The identity of PrP interacting proteins was investigated during the purification of PrP under differential extraction conditions in which prion infectivity was observed using an in vitro assay. Proteinase K digestion gave a change in prion infectivity implying that a correlation with the removal of a protease sensitive component occurred. Continuation of these studies may facilitate the identification of co-factors required for specific prion infectivity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.439301  DOI: Not available
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