NR AJJE
AU Pergami,P.; Jaffe,H.; Safar,J.G.
TI Semipreparative chromatographic method to purify the normal cellular isoform of the prion protein in nondenatured form
QU Analytical Biochemistry 1996 Apr 5; 236(1): 63-73
PT journal article
AB 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.
IN Die Autoren entwickelten eine Methode, das normale Prionprotein aus Hamsterhirnen anscheinend ohne Strukturveränderung mit akzeptablen Verlusten sehr stark anzureichern. Zunächst werden die Membranen von Synapsen und Mikrosomen isoliert. Daraus werden die Prionproteine mit dem nichtionischen Detergens n-Octyl-beta-Glucopyranosid gelöst und bei pH 7,5 auf eine semipräparative TSK-SP-BPW (HPLC)-Kationaustauschersäule geladen. Die mit linear gesteigerten NaCl-Konzentrationen eluierten Fraktionen mit den größten Prionproteinkonzentrationen werden anschließend auf einer mit Co2+ geladenen Ionenaffinitäts-HPLC-Säule und danach mit einer Weizenkeimagglutinin-Affinitäts-HPLC oder Größenausschluß-HPLC (TSK G3000SW) gereinigt. Noch dieser Prozedur sollen noch mindestens 8% des normalen Prionproteins in einer Reinheit von 95% übrigbleiben. Die gereinigten zellulären Prionproteine liegen als intakte Monomere mit einem Stoke's-Radius von 26 Angström vor und zeigen ein normales Trypsin-Spaltmuster.
ZR 40
MH Amino Acid Sequence; Animal; Brain Chemistry; Chromatography, Affinity/methods; Chromatography, High Pressure Liquid/*methods; Detergents; Glucosides/chemistry; Guanidine; Guanidines; Hamsters; Mesocricetus; Microsomes/chemistry; Molecular Sequence Data; Peptide Mapping; Prions/*isolation & purification; Protein Denaturation; Protein Structure, Secondary; Synaptosomes/chemistry
AD Paola Pergami, Jiri Safar, Laboratory of Central Nervous System Studies, National Institutes of Health, Bldg. 36, Room 4A-05, Bethesda, Maryland, 20892-4122, USA; Howard Jaffe, Division of Intramural Research, Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
SP englisch
PO USA