Purification and properties of the cellular prion protein from Syrian hamster brain

Abstract

The cellular prion protein (PrP^C) is encoded by a chromosomal gene, and its scrapie isoform (PrP^Sc) features in all aspects of the prion diseases. Prior to the studies reported here, purification of PrP^C has only been accomplished using immunoaffinity chromatography yielding small amounts of protein. Brain homogenates contain two PrP^C forms designated PrP^C-I and -II. These proteins were purified from a microsomal fraction by detergent extraction and separated by immobilized Cu²⁺ ion affinity chromatography. PrP^C-II appears to be generated from PrP^C-I by limited proteolysis of the N-terminus. Fractions enriched for PrP^C-I were purified further by cation-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Greater than 90% of the final product migrated as a broad band of M_r 33–35 kDa as judged by silver staining after SDS-PAGE. Digestion of PrP^C-I with peptide-N-glycosidase (PNGase) compressed the band and shifted its mobility giving an M_r of 27 kDa. The protocol described should be amenable to large-scale preparation of PrP^C, enabling physical comparisons of PrP^C and PrP^Sc.