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-5PW (HPLC) column. The fractions 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 >/=8%. The purified PrPC was a monomer with an intact N-terminus, and with a Stoke's radius of 26 A, 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.
Pergami
P
P
Laboratory of Central Nervous System Studies, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Jaffe
H
H
Safar
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D003902:Detergents; D005960:Glucosides / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines; D008647:Mesocricetus; D008861:Microsomes / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011328:Prions / Q000302:isolation & purification*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013574:Synaptosomes / Q000737:chemistry
D003902:Detergents; D005960:Glucosides; D006146:Guanidines; D011328:Prions; C018619:octyl-beta-D-glucoside; D019791:Guanidine