Purification and properties of the cellular prion protein from Syrian hamster brain.
The cellular prion protein (PrPC) is encoded by a chromosomal gene, and its scrapie isoform (PrPSc) features in all aspects of the prion diseases. Prior to the studies reported here, purification of PrPC has only been accomplished using immunoaffinity chromatography yielding small amounts of protein. Brain homogenates contain two PrPC forms designated PrPC-I and -II. These proteins were purified from a microsomal fraction by detergent extraction and separated by immobilized Cu2+ ion affinity chromatography. PrPC-II appears to be generated from PrPC-I by limited proteolysis of the N-terminus. Fractions enriched for PrPC-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 PrPC-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 PrPC, enabling physical comparisons of PrPC and PrPSc.
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Department of Neurology, University of California, San Francisco 94143.
Stahl
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Prusiner
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D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003902:Detergents; D006031:Glycosylation; D008647:Mesocricetus; D008861:Microsomes / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018620:PrPSc Proteins; D011328:Prions / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D009419:Nerve Tissue Proteins; D018620:PrPSc Proteins; D011328:Prions