The Development of a Quantitative Assay to Measure the Effects of Protein Disulfide Isomerase on Prion Protein Misfolding

Abstract

The objective ofthis project was to develop a quantitative assay to measure the misfolding ofprion proteins that can be used to assess the effects of Protein Disulfide Isomerase (PDI) on the misfolding process ofthe Chronic Wasting Disease (CWD) prion protein. CWD is a spongiform encephalopathy native to animals in the family Cervidae, which includes whitetail deer, mule deer, elk and moose. CWD is caused by the misfolding ofprion proteins. Qualitative observations from previous studies indicate that disulfide bond rearrangement might be a rate limiting step in the misfolding process. There is a need to be able to quantitate the amount of misfolded prion protein, and this was achieved through a limiting dilution, slot blot analysis using a Bio Dot® SF Microfiltration apparatus. The limiting dilution, slot blot analysis was standardized parallel with conventional immunoblotting and was optimized using differing buffers and immunoblot membranes. This quantitative technique can be used to measure the effect of PDI on the misfolding of CWD prion proteins.