title: In vitro cell-free conversion of bacterial recombinant PrP to PrPres as a model for conversion.
abstract: Prion diseases are associated with the conversion of the normal cellular prion protein, PrP(C), to the abnormal disease-associated protein, PrP(Sc). This conversion can be mimicked in vitro using PrP(Sc) isolated from the brains of scrapie-infected animals to induce conversion of recombinant PrP(C) into a proteinase K-resistant isoform, PrP(res). Traditionally, the 'cell-free' conversion assay has used, as substrate, recombinant PrP(C) purified from mammalian tissue culture cells or, more recently, from baculovirus-infected insect cells. The cell-free conversion assay has been modified by replacing the tissue culture-derived PrP(C) with recombinant PrP purified from bacteria. Bacterial expression and chromatographic purification give high yields of recombinant radiolabelled untagged protein, eliminates artefacts that may be due to cellular factors or antibody fragments normally present in labelled PrP preparations and allows accurate and rapid variation of protein sequence using standard molecular biological techniques. In addition, these cell-free conversion assays were carried out under more physiological conditions, giving more relevance to the assay as a model for conversion. To validate its use in this assay, this bacterial recombinant PrP has been shown to have the conversion properties of mammalian PrP(C): (i) it converts to a proteinase K-resistant isoform in the presence of PrP(Sc); (ii) the efficiency of this conversion by PrP(Sc) of different strains and species parallels that found in vivo; and (iii) its cell-free conversion is inhibited by Congo Red analogues in a structure-dependent manner similar to that seen in in vivo and in vitro cell assays.
authors.affiliation: Institute for Animal Health, Compton Laboratories, Newbury, Berkshire RG20 7NN, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Kirby
authors.affiliation: Institute for Animal Health, Compton Laboratories, Newbury, Berkshire RG20 7NN, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Birkett
authors.affiliation: Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK.
authors.forename: Helene
authors.initials: H
authors.lastname: Rudyk
authors.affiliation: Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK.
authors.forename: Ian H
authors.initials: IH
authors.lastname: Gilbert
authors.affiliation: Institute for Animal Health, Compton Laboratories, Newbury, Berkshire RG20 7NN, UK.
authors.forename: James
authors.initials: J
authors.lastname: Hope
mesh_terms: D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D006224:Cricetinae; D019286:Endopeptidase K / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D051379:Mice; D018621:PrPC Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018620:PrPSc Proteins / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D018621:PrPC Proteins; D018620:PrPSc Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019286:Endopeptidase K