Isolation and structural studies of the intact scrapie agent protein

Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27–30, with an apparent mass of 27–30 kDa (D. C. Bolton et al. (1982) Science 218, 1309–1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942–6950). In contrast, a 33–37 kDa glycoprotein, HaSp33–37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33–37 had >10 11 LD 50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33–37 gave a product indistinguishable from PrP-27–30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33–37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27–30 (K. Basler et al. (1986) Cell 46, 417–428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377–6381) after processing by signal protease. HaSp33–37 was digested with N α-tosyl- l-phenylalanine chloromethyl ketone-trypsin to produce a 29–32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29–32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33–37 and PrP-27–30. We conclude that HaSp33–37 is the intact form of the scrapie agent protein and that PrP-27–30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.