Semipreparative chromatographic method to purify the normal cellular isoform of the prion protein in nondenatured form

A fundamental step in the pathogenesis of spongiform encephalopathies (prion diseases) is the conversion of the cellular isoform of prion protein (PrPC) into the infectious form (scrapie isoform, PrPSc), apparently by a conformational mechanism, Comparison of the native secondary and tertiary structures of both proteins is essential to elucidate the molecular basis of this transformation, To obtain sufficient quantities of native-like PrPC, we have developed a semipreparative method to purify PrPC from hamster brains, PrPC was solubilized from purified synaptosomal and microsomal membranes by the nonionic detergent n-octyl-beta-glucopyranoside; the soluble fraction was loaded at pH 7.5 onto a semipreparative cation-exchange TSK-SP-BPW (HPLC) column, The fr actions eluted by linear NaCl gradient and enriched for PrPC were sequentially purified using an immobilized ion-affinity HPLC column charged by Co2+, followed by wheat germ agglutinin (WGA)-affinity HPLC or size-exclusion HPLC (SE-HPLC) using a TSK G3000SW column, More than 95% purity was achieved after SE-HPLC as estimated by quantitative densitometry of the silver-stained SDS-PAGE gel; the recovery of total brain PrPC was greater than or equal to 8%. The purified PrPC was a monomer with an intact N-terminus, and with a Stoke's radius of 26 Angstrom, corresponding to that expected from the molecular weight for a native protein, The presence of the native-like conformation was further verified by peptide mapping after limited trypsin proteolysis, and by the apparent unfolding in guanidine hydrochloride, as detected by SE-HPLC. (C) 1906 Academic Press, Inc.