Molecular Biotechnology

, Volume 35, Issue 2, pp 161–170 | Cite as

Use of thermolysin in the diagnosis of prion diseases

  • Jonathan P. Owen
  • Ben C. Maddison
  • Garry C. Whitelam
  • Kevin C. Gough


The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPc into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27–30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.

Index Entries

PrP prion protein scrapie BSE thermolysin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Prusiner, S. B. (1998) Prions, Proc. Natl. Acad. Sci. USA 95, 13363–13383.PubMedCrossRefGoogle Scholar
  2. 2.
    van Rheede, T., Smolenaars, M. M. W., Madsen, O., and de Jong W.W. (2003). Molecular evolution of the mammalian prion protein. Mol. Biol. Evol. 20, 111–121.PubMedCrossRefGoogle Scholar
  3. 3.
    Sakudo, A., Lee, D., Nishimura, T., et al. (2005) Octopeptide repeat region and N-terminal half of hydrophobic region of prion protein (PrP) mediate PrP-dependent activation of superoxide dismutase. Biochem. Biophys. Res. Commun. 326, 600–606.PubMedCrossRefGoogle Scholar
  4. 4.
    Moynagh, J. and Schimmel, H. (1999) Tests for BSE evaluated. Nature 400, 105.PubMedCrossRefGoogle Scholar
  5. 5.
    Schaller, O., Fatzer, R., Stack, M., et al. (1999) Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol (Berl.) 98, 437–443.CrossRefGoogle Scholar
  6. 6.
    Grassi, J., Comoy, E., Simon, S., et al. (2001) Rapid test for the preclinical postmortem diagnosis of BSE in central nervous system tissue. Vet. Rec. 149, 577–582.PubMedGoogle Scholar
  7. 7.
    Biffiger, K., Zwald, D., Kaufmann, L., et al. (2002) Validation of a luminescence immunoassay for the detection of PrPSc in brain homogenate. J. Virol. Methods. 101, 79–84.PubMedCrossRefGoogle Scholar
  8. 8.
    Safar, J. G., Scott, M., Monaghan, J., et al. (2002) Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice. Nature Biotechnol. 20, 1147–1150.CrossRefGoogle Scholar
  9. 9.
    Todorova-Balvay, D., Simon, S., Creminon, C., Grassi, J., Srikrishnan, T., and Vijayalakshmi, M. A. (2005) Copper binding to prion protein octarepeat peptides, a combined metal chelate affinity and immunochemical approach. J. Chromat. 818, 75–82.CrossRefGoogle Scholar
  10. 10.
    Feraudet, C., Morel, N., Simon, S., et al. (2005) Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells. J. Biol. Chem. 280, 11247–11258.PubMedCrossRefGoogle Scholar
  11. 11.
    Oesch, B., Westaway, D., Wälchli, M., et al. (1985) A cellular gene encodes scrapie PrP 27–30 protein. Cell 40, 735–746.PubMedCrossRefGoogle Scholar
  12. 12.
    Parchi, P., Zou, W. Q., Wang, W., et al. (2000) Genetic influence on the structural variations of the abnormal prion protein. Proc. Natl. Acad. Sci. USA 97, 10168–10172.PubMedCrossRefGoogle Scholar
  13. 13.
    Harmeyer, S., Pfaff, E. and Groschup M. H. (1998) Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants. J. Gen. Virol. 79, 937–945.PubMedGoogle Scholar
  14. 14.
    Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R., and Cesareni, G. (1991) Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 222, 301–310.PubMedCrossRefGoogle Scholar
  15. 15.
    Felici, F., Luzzago, A., Folgori, A., and Cortese, R. (1993) Mimicking of discontinuous epitopes by phagedisplayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries. Gene 128, 21–27.PubMedCrossRefGoogle Scholar
  16. 16.
    Vincent, B., Paitel, E., Froberts, Y., Lehmann, S., Grassi, J., and Checler, F. (2000) Phorbol ester-regulated cleavage of normal prion protein in HEK293 human calls and murine neurons. J. Biol. Chem. 275, 35612–35616.PubMedCrossRefGoogle Scholar
  17. 17.
    Arnold, U., Rücknagel, K. P., Schierborn, A., and Ulbrich-Hofmann, R. (1996) Thermal unfolding and proteolytic susceptibility of ribonuclease A. Eur. J. Biochem. 237, 862–869.PubMedCrossRefGoogle Scholar
  18. 18.
    Bark, S. J., Muster, N., Yates III, J. R., and Siuzdak, G. (2001) High-temperature protein mass mapping using a thermophylic protease. J. Am. Chem. Soc. 123, 1774–1775.PubMedCrossRefGoogle Scholar
  19. 19.
    Hope J., Wood, S. C. E. R., Birkett, C. R., et al. (1999) Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J. Gen. Virol. 80, 1–4.PubMedGoogle Scholar
  20. 20.
    Wadsworth, J. D. F., Joiner, S., Hill, A. F., et al. (2001). Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 358, 171–180.PubMedCrossRefGoogle Scholar
  21. 21.
    Frankenfield, K. N., Powers, E. T., and Kelly, J. W. (2005) Influence of the N-terminal domain on the aggregation properties of the prion protein. Protein Sci. 14, 2154–2166.PubMedCrossRefGoogle Scholar
  22. 22.
    Bocharova, O. V., Breydo, L., Salnikova, V. V. and Baskakov, I. V. (2005) Copper (II) inhibits in vitro conversion of prion protein into amyloid fibrils. Biochem. 44, 6776–6787.CrossRefGoogle Scholar
  23. 23.
    Li, R., Liu, T., Wong, B. S., et al. (2000) Identification of an epitope in the C-terminus of normal prion protein whose expression is modulated by binding events at the N-terminus. J. Mol. Biol. 301, 567–573.PubMedCrossRefGoogle Scholar
  24. 24.
    Silveira, J. R., Raymond, G. J., Hughson, A. G., et al. (2005) The most infectious prion protein particles. Nature 437, 257–261.PubMedCrossRefGoogle Scholar
  25. 25.
    Pan, T., Wong, P., Chang, B., et al (2005) Biochemical fingerprints of prion infection: accumulation of aberrant full-length and N-terminally truncated PrP species are common features in mouse prion disease. J. Virol. 79, 934–943.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Jonathan P. Owen
    • 1
  • Ben C. Maddison
    • 1
  • Garry C. Whitelam
    • 1
  • Kevin C. Gough
    • 1
  1. 1.ADAS UK, Department of Biology, Adrian BuildingUniversity of LeicesterLeicesterUK

Personalised recommendations