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|Title:||Scrapie associated fibrils and polymeric PrP|
|Authors:||Potter, Richard J.|
|Presented at:||University of Leicester|
|Abstract:||The conversion of PrPC, the normal form of the prion protein, to PrPSc, the protease-resistant disease specific form, is central to the pathogenesis of the transmissible spongiform encephalopathies. The mechanism underlying this conformational change remains elusive but the demonstration that PrPSc can infer protease-resistance to PrPc in-vitro in the presence of guanidine hydrochloride using a cell-free system offers a useful approach to investigating this process. The limitations of such systems however are that significant efficiencies of conversion are only observed in the presence of an excess of PrPSc which precludes both the detection of new infectivity by bioassay and investigation of the mechanism involved.;To investigate these observations recombinant murine PrP (rMoPrP) expressed in E. coli was converted to a protease-resistant form in the presence of a equal amount of MoPrPSc using a similar approach. The reaction involves a specific interaction between the two PrP species culminating in the generation of conversion products that are highly resistant to proteolysis. The largest conversion reaction product has a mass of ~17 kda and maps to residues ~90-231 of full length MoPrP which equates to PrP27-30, the protease resistant core of PrPSc.;Recombinant MoPrP, tagged with the hamster specific epitope to the monoclonal antibody 3F2 (rMoPrP(3F4)), was converted to a protease resistance form, rMoPrP(3F4)res, using fibrillar MoPrP27-30. Sequential conversions were attempted and the level of rMoPrP(3F4)res present following each reaction was found to be cumulative. When examined by immuno-gold electron microscopy using 3F4 no labelling of the fibrils could be detected in these fractions but amorphous material associated with these structures was heavily labelled as observed for fibrillar hamster PrP27-30 indicating that rPrP had been converted to a form resembling that present in infectious fractions. This study provides data showing an excess of converted recombinant PrP (rMoPrP(3F4)res) over 'seeding-PrP' (PrP27-30) which may ultimately facilitate the detection of infectivity generated in-vitro.;.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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