Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis
A) A misfolded, propagative mouse PrP species (rec-MoPrPSc-950) was generated in the presence of lipids and RNAusing RecMo-PrPSc-17KDa, generated in Grand Rapids (MI, USA), as seed in a recPMCA reaction. The seed was diluted 1:10 in each round of recPMCA up to 17 rounds and the resulting product was digested with 10 μg/ml of PK. The final product is termed “Sc” as its infectivity in vivo was later confirmed (Fig 2). B) A misfolded, propagative bank vole PrP species (recBVPrPSc) was generated utilizing a different version of seeded recPMCA (details to be described elsewhere), using recBVPrP23-231 PrP as a substrate. Twenty five rounds were completed. The resulting product was digested with 85 μg/ml of PK. The final sample is termed “Sc” as its infectivity in vivo was later confirmed (see Results section). Both samples were analyzed by Western blot using monoclonal antibody Saf83 (1:400). Undigested bank vole and mouse rec-PrP were used as control. MW: molecular weight markers. In A, central lanes with irrelevant samples have been removed from the image for clarity.