Progress in studies on the structural transition of PrPc into PrPSc requires soluble PrPSc. Toward this goal, we propose to exploit an exciting set of findings where approximately 50% of the PrP molecule was deleted and yet the mutant protein could be converted into PrPSc. This mutant PrP contains 106 residues and is denoted PrP106 while the pathogenic isoform is designated PrPS106. In scrapie infected mouse neuroblastoma (ScN2a) cells, PrPSc106 is protease-resistant and can be solubilized in 1% Sarkosyl under conditions that are known to preserve prion infectivity. Furthermore, Tg (PrP106)Prnp0/0 mice developed signs of CNS dysfunction at approximately 65 days after inoculation with RML106 prions. We propose to characterize prions containing PrPSc106, to develop suitable large-scale purification procedures, and to perform structural studies of PrPSc106. Standard curves for bioassay of RML106 prions will be constructed. Optimal conditions for solubilizing PrPSc106 from transfected ScN2a cells, brains of Tg(PrP106) Prnp0/0 mice and cultured cells derived from embryos PrPSc106, we shall identify suitable hydrophobic residues on the surface of PrP106 that can be substituted with hydrophilic residues. A combinatorial strategy will be employed to mutate several sites simultaneously, and a screening protocol using an automated robotic workstation employed. N- terminally modified PrP106 will be tested for increasing the rate of PrPSc106 formation and elevating the levels of this pathogenic isoform. The charged N-terminal residues will be used as an epitope for generating anion- dependent antibodies that could be used in a high yield, affinity purification scheme. Multiple strains of PrP106 prions will be produced by passage in Tg(PrP106)Prnp0/0 mice if the physical properties of PrPSc106 from any strain are more amenable to physical characterization than those of PrPSc106 from RML106.
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