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Purification of the Scrapie Agent by Density Gradient Centrifugation

Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin 53706, U.S.A.

Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains, as well as preparations of neural retina, were sonicated, then separated by rate-zonal sedimentation in 10 to 25% Nycodenz® gradients. Gradient fractions were extracted with 0.5% Triton X-100 and re-fractionated by equilibrium density centrifugation in linear 25 to 40% CsCl gradients. Infectivity was highest in a fraction having a density of 1.280 g/ml and which contained a visible band of material. Digestion of the Nycodenz fractions with proteinase K before detergent extraction and CsCl fractionation resulted in a shift in the visible band to a density of 1.235 g/ml with most of the scrapie infectivity remaining at 1.280 g/ml. When labelled with ¹²⁵I after 40-fold concentration, this 1.280 g/ml CsCl fraction from the proteinase K-treated gradients contained only a single band of protein(s) having a mol. wt. near 30000. No differences were seen between proteins in healthy or scrapie-infected preparations.

Keywords: scrapie, gradient centrifugation, membrane vesicle, protein characterization

Received 9 August 1983; accepted 7 November 1983.