Advertisement

Synthesis of Peptide Sequences Derived from Fibril-Forming Proteins

  • Denis B. Scanlon
  • John A. Karas
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 752)

Abstract

The pathogenesis of a large number of diseases, including Alzheimer’s Disease, Parkinson’s Disease, and Creutzfeldt–Jakob Disease (CJD), is associated with protein aggregation and the formation of amyloid, fibrillar deposits. Peptide fragments of amyloid-forming proteins have been found to form fibrils in their own right and have become important tools for unlocking the mechanism of amyloid fibril formation and the pathogenesis of amyloid diseases. The synthesis and purification of peptide sequences derived from amyloid fibril-forming proteins can be extremely challenging. The synthesis may not proceed well, generating a very low quality crude product which can be difficult to purify. Even clean crude peptides can be difficult to purify, as they are often insoluble or form fibrils rapidly in solution. This chapter presents methods to recognise and to overcome the difficulties associated with the synthesis, and purification of fibril-forming peptides, illustrating the points with three synthetic examples.

Key words

Solid-phase peptide synthesis Fibril-forming peptide Microwave peptide synthesis Peptide HPLC purification ApoCII [56–76] Aβ[1–42] Prion protein 

Notes

Acknowledgements

We thank Ms Keyla Perez for her advice with the Vydac C4 preparative column technology and peptide purifications performed at 60°C.

References

  1. 1.
    Merrifield, R. B. (1963) Solid Phase Synthesis I. The Synthesis of a Tetrapeptide. J. Amer. Chem. Soc. 85, 2149–2154.CrossRefGoogle Scholar
  2. 2.
    Kent, S. B., Mitchell, A. R., Engelhard, M. and Merrifield, R. B.(1979) Mechanisms and prevention of trifluoroacetylation in solid-phase peptide synthesis. Proc. Natl. Acad. Sci. U S A. 76(5), 21802184.PubMedCrossRefGoogle Scholar
  3. 3.
    Sarin, V. K., Kent, S. B., Tam, J. P. and Merrifield, R. B. (1981) Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction Anal. Biochem. 117(1), 147157.Google Scholar
  4. 4.
    Brown, E., Sheppard, R. C. and Williams, B. J. (1983) Peptide Synthesis. Part 5. Solid-phase Synthesis of [15-Leucine] Little Gastrin. J. Chem. Soc. Perkin Trans. I, 1161–1167.Google Scholar
  5. 5.
    Sheppard, R. C. (1986) Modern methods of solid-phase peptide synthesis. Science Tools 33, 9–16.Google Scholar
  6. 6.
    Schnolzer, M., Alewood, P., Jones, A., Alewood, D. and Kent, S. B. H. (1992) In situ neutralization in Boc-chemistry solid phase peptide synthesis. Int. J. Peptide Protein Res. 40, 180–193.CrossRefGoogle Scholar
  7. 7.
    Alberico, F. and Carpino, L. A. (1997) Coupling reagents and activation Methods Enzymol. 289, 104–126.Google Scholar
  8. 8.
    Clark-Lewis, I., Aebersold, R., Ziltener, H., Schrader, J. W., Hood, L. E. and Kent, S. B. H. (1986) Automated Chemical Synthesis of a Protein Growth Factor for Hemopoietic Cells, Interleukin-3. Science 231, 134–139.PubMedCrossRefGoogle Scholar
  9. 9.
    Scanlon, D. B., Eefting, M. A., Lloyd, C. J., Burgess, A. W. and Simpson, R. J. (1987) Synthesis of Biologically Active Transforming Growth Factor-alpha by Fluorenyl­methoxy­carbonyl Solid Phase Peptide Chemistry. J. Chem. Soc. Chem. Commun. 516–518.Google Scholar
  10. 10.
    Dawson, P. E., Muir, T. W., Clark-Lewis, I. and Kent, S. B. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776779.PubMedCrossRefGoogle Scholar
  11. 11.
    Yamamoto, N., Tanabe, Y., Okamoto, R., Dawson, P.E. and Kajihara, Y. (2008) Chemical Synthesis of a Glycoprotein Having an Intact Human Complex-Type Sialyloligosaccharide under the Boc and Fmoc Synthetic Strategies J. Am. Chem. Soc. 130(2), 501–510.CrossRefGoogle Scholar
  12. 12.
    Macmillan, D. (2006) Protein Synthesis: Evolving Strategies for Protein Synthesis Converge on Native Chemical Ligation Angew. Chem. Int. Ed. 45, 7668–7672.CrossRefGoogle Scholar
  13. 13.
    Tickler, A. K.,. Clippingdale, A. B. and Wade, J. D. (2004) Amyloid-β as a “Difficult Sequence” in Solid Phase Peptide Synthesis. Protein & Peptide Letters 11(4), 377–384.CrossRefGoogle Scholar
  14. 14.
    Mutter, M., Nefzi, A., Sato, T., Sun, X., Wahl, F., and Wohr, T. (1995) Pseudo-prolines for accessing “inaccessible” peptides Peptide Research 8(3), 145–153.PubMedGoogle Scholar
  15. 15.
    Simmonds, R. G. (1996) Use of the Hmb backbone-protecting group in the synthesis of difficult sequences. Int. J. Pept. Protein Res. 47(1–2), 36–41.PubMedGoogle Scholar
  16. 16.
    Tickler, A. K., Barrow, C. J. and Wade, J. D. (2001) Improved Preparation of Amyloid-Peptides Using DBU as N-Fmoc Deprotection Reagent J. Peptide Sci. 7, 488–494.CrossRefGoogle Scholar
  17. 17.
    Sohma, Y. and Kiso, Y. (2006) “Click peptides”-chemical biology-oriented synthesis of Alzheimer’s disease-related amyloid beta peptide (abeta) analogues based on the “O-acyl isopeptide method”. Chembiochem. 7(10), 15491557.PubMedCrossRefGoogle Scholar
  18. 18.
    Taniguchi, A., Sohma, Y., Hirayama, Y., Mukai, H., Kimura, T., Hayashi, Y., Matsuzaki, K., Kiso, Y. (2009) “Click peptide”: pH-triggered in situ production and aggregation of monomer Abeta1-42. Chembiochem. 10(4), 710715.PubMedCrossRefGoogle Scholar
  19. 19.
    Howlett, G. J., and Moore, K. J. (2006) Untangling the role of amyloid in atherosclerosis Current Opinion in Lipidology 17, 541–547.CrossRefGoogle Scholar
  20. 20.
    Wilson, L. M., Mok, Y. F., Binger, K. J., Griffin, M. D., Mertens, H. D., Lin, F., Wade, J. D., Gooley, P. R. and Howlett, G. J. (2007) A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis. J. Mol. Biol. 366(5), 16391651.PubMedCrossRefGoogle Scholar
  21. 21.
    Van Nostrand, W.E., Davis-Salinas, J. and Saporito-Irwin, S. M. (1996) Amyloid beta-protein induces the cerebrovascular cellular pathology of Alzheimer’s disease and related disorders Ann. N Y. Acad. Sci. 777, 297–302.Google Scholar
  22. 22.
    Ball, H. L. and Mascagni, P. (1996) Chemical protein synthesis and purification: a methodology. Int. J. Peptide Protein Res. 48,31–47.CrossRefGoogle Scholar
  23. 23.
    Bonetto, V., Massignan, T., Chiesa, R., Morbin, M., Mazzoleni, G., Diomede, L., Angeretti, N., Colombo, L., Forloni, G., Tagliavini, F., and Salmona, M. (2002) Synthetic miniprion PrP106 J. Biol. Chem. 277, 31327–31334.CrossRefGoogle Scholar
  24. 24.
    Bahadi, R., Farrelly, P. V., Kenna, B. L., Kourie, J. I., Tagliavini, F., Forloni, G. and Salmona, M. (2003) PrP(82–146) homologous to a 7-kDa fragment in Channels formed with a mutant prion protein diseased brain of GSS patients Am. J. Physiol. Cell. Physiol. 285, 862872.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneParkvilleAustralia

Personalised recommendations