title: Isolation of proteinase K-sensitive prions using pronase E and phosphotungstic acid.
abstract: Disease-related prion protein, PrP(Sc), is classically distinguished from its normal cellular precursor, PrP(C), by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrP(Sc) using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrP(C), while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrP(C).
authors.affiliation: MRC Prion Unit and Department of Neurodegenerative Disease, UCL Institute of Neurology, National Hospital for Neurology and Neurosurgery, London, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: D'Castro
authors.forename: Adam
authors.initials: A
authors.lastname: Wenborn
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gros
authors.forename: Susan
authors.initials: S
authors.lastname: Joiner
authors.forename: Sabrina
authors.initials: S
authors.lastname: Cronier
authors.forename: John
authors.initials: J
authors.lastname: Collinge
authors.forename: Jonathan D F
authors.initials: JD
authors.lastname: Wadsworth
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D051379:Mice; D010772:Phosphotungstic Acid / Q000378:metabolism*; D018620:PrPSc Proteins / Q000378:metabolism; D017096:Prion Diseases / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism*; D012608:Scrapie / Q000378:metabolism; D016622:Silver Staining
chemical_list: D018620:PrPSc Proteins; D011328:Prions; D010772:Phosphotungstic Acid; D019286:Endopeptidase K; D011402:Pronase