Developing a Method for In Vitro Conversion of Recombinant Mule Deer Prion Protein

Abstract

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) occurring in free-ranging and captive populations of deer and elk. CWD is thought to involve a misfolded protein called the prion protein. The misfolding SC mechanism remains unknown, but the conversion results from misfolded PrP contacting normal PrPc, and inducing a conformational change of PrPcto PrPsc. Previous studies have shown the normal cellular PrPc, isolated from mule deer brain, has the ability to misfold in vitro when incubated with misfolded PrPsc. Instead of using deer brain as a source of the normal cellular form of the prion protein, this project examined the use of recombinant mule deer prion protein expressed in High 5 insect cells. The cells were infected with a recombinant baculovirus that encodes and expresses the recombinant mule deer prion protein, rPrPc. Developing in vitro conversion methods for the recombinant form of the prion protein will enable future studies that use this substrate to probe the mechanism of the misfolding process. Two methods, nondenaturing and protein-misfolding-cyclic-amplification (PMCA), have been devised to study the misfolding process in vitro. The observations reported here suggest that the recombinant mule deer prion protein was converted to the misfolded isoform by both nondenaturing and PMCA methodologies.