HPLC Methods for Purification of Antimicrobial Peptides

  • Michael E. Selsted
Part of the Methods In Molecular Biology™ book series (MIMB, volume 78)


The advent of high performance liquid chromatography (HPLC) has greatly accelerated the discovery, purification, and characterization of antimicrobial peptides. Virtually every modern study of an antimicrobial peptide includes or was preceded by a description of its purification. The increased pace of peptide discovery and characterization has resulted from the development of sophisticated column and solvent delivery technology over the last four decades. Interestingly, the modern methods described here derive directly from standard open-column (“low performance”) chromatographic modalities. Therefore, it is not surprising that virtually every method used in traditional column chromatography has been adapted to high performance methods. These include gel filtration, ion-exchange, and reversed-phase chromatography methods described in this chapter.


High Performance Liquid Chromatography High Performance Liquid Chromatography Antimicrobial Peptide High Performance Liquid Chromatography Method High Performance Liquid Chromatography System 
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  1. 1.
    Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 62–92.CrossRefGoogle Scholar
  2. 2.
    Martin, E., Ganz, T., and Lehrer, R. I. (1995) Defensins and other endogenous peptide antibiotics of vertebrates. J. Leukocyte Biol. 58, 128–136.PubMedGoogle Scholar
  3. 3.
    Lehrer, R. I., Lichtenstein, A. K., and Ganz, T. (1993) Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu. Rev. Immunol. 11, 105–128.PubMedCrossRefGoogle Scholar
  4. 4.
    Ouellette, A. J., Hsieh, M. M., Nosek, M. T., Cano-Gauci, D. F., Huttner, K. M., Buick, R. N., and Selsted, M. E. (1994) Mouse paneth cell defensins primary structures and antibacterial activities of numerous cryptdin isoforms. Infect. Immun. 62, 5040–5047.PubMedGoogle Scholar
  5. 5.
    Selsted, M. E. and Ouellette, A. J. (1995) Defensins in granules of phagocytic and non-phagocytic cells. Trends in Cell Biology 5, 114–119.PubMedCrossRefGoogle Scholar
  6. 6.
    Jones, D. E. and Bevins, C. L. (1992) Paneth cells of the human small intestine express an antimicrobial peptide gene. J. Biol. Chem. 267, 23,216–23,225.PubMedGoogle Scholar
  7. 7.
    Jones, D. E. and Bevins, C. L. (1993) Defensin-6 mRNA in human Paneth cells: implications for antimicrobial peptides in host defense of the human bowel. FEBS Lett. 315, 187–192.PubMedCrossRefGoogle Scholar
  8. 8.
    Selsted, M. E., Tang, Y.-Q., Morris, W. L., McGuire, P. A., Novotny, M. J., Smith, W., Henschen, A. H., and Cullor, J. S. (1993) Purification, primary structures, and antibacterial activities of β-defensins, a new family of antimicrobial peptides from bovine neutrophils. J. Biol. Chem. 268, 6641–6648.PubMedGoogle Scholar
  9. 9.
    Diamond, G., Zasloff, M., Eck, H., Brasseur, M., Maloy, W. L., and Bevins, C. L. (1991) Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc. Natl. Acad. Sci. USA 88, 3952–3956.PubMedCrossRefGoogle Scholar
  10. 10.
    Schonwetter, B. S., Stolzenberg, E. D., and Zasloff, M. A. (1995) Epithelial antibiotics induced at sites of inflammation. Science 267, 1645–1648.PubMedCrossRefGoogle Scholar
  11. 11.
    Bensch, K. W., Raida, M., Magert, H.-J., Schulz-Knappe, P., and Forssmann, W.-G. (1995) hBD-1: a novel β-defensin from human plasma. FEBS Lett. 368, 331–335.PubMedCrossRefGoogle Scholar
  12. 12.
    Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.-Y., and Lidholm, D.-A. (1991) Cell-free immunity in Cecropia. A model system for antibacterial proteins. Eur. J. Biochem. 201, 23–31.PubMedCrossRefGoogle Scholar
  13. 13.
    Bevins, C. L. and Zasloff, M. (1990) Peptides from frog skin. Ann. Rev. Biochem. 59, 395–414.PubMedCrossRefGoogle Scholar
  14. 14.
    Hoffmann, J. A. and Hetru, C. (1992) Insect defensins, inducible antibacterial peptides. Immunol. Today 13, 411–415.PubMedCrossRefGoogle Scholar
  15. 15.
    Broekaert, W. F., Terras, F. R. G., Cammue, B. P. A., and Osborn, R. W. (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol. 108, 1353–1358.PubMedCrossRefGoogle Scholar
  16. 16.
    Selsted, M. E., Novotny, M. J., Morris, W. L., Tang, Y.-Q., Smith, W., and Cullor, J. S. (1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J. Biol. Chem. 267, 4292–4295.PubMedGoogle Scholar
  17. 17.
    Selsted, M. E., Miller, S. I., Henschen, A. H., and Ouellette, A. J. (1992) Enteric defensins: antibiotic peptide components of intestinal host defense. J. Cell Biol. 118, 929–936.PubMedCrossRefGoogle Scholar
  18. 18.
    Harwig, S. S. L., Ganz, T., and Lehrer, R. I. (1994) Neutrophil defensins purification, characterization, and antimicrobial testing. Meth. in Enzymol. 236, 160–172.CrossRefGoogle Scholar
  19. 19.
    Selsted, M. E., Szklarek, D., and Lehrer, R. I. (1984) Purification and antibacterial activity of antimicrobial peptides of rabbit granulocytes. Infect. Immun. 45, 150–154.PubMedGoogle Scholar
  20. 20.
    Lichtenstein, A., Ganz, T., Selsted, M. E., and Lehrer, R. I. (1986) In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes. Blood 68, 1407–1410.PubMedGoogle Scholar
  21. 21.
    Zasloff, M. (1987) Magainins, a class of antimicrobial peptides from Xenopus skin, isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc. Nat. Acad. Sci. USA 84, 5449–5453.PubMedCrossRefGoogle Scholar
  22. 22.
    Gennaro, R., Skerlavaj, B., and Romeo, D. (1989) Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils. Infect. Immun. 57, 3142–3146.PubMedGoogle Scholar
  23. 23.
    Lee, J.-Y., Boman, A., Chuanxin, S., Andersson, M., Jornvall, H., Mutt, V., and Boman, H. G. (1989) Antibacterial peptides from pig intestine: Isolation of a mammalian cecropin. Proc. Nat. Acad. Sci. USA 86, 9159–9162.PubMedCrossRefGoogle Scholar
  24. 24.
    Agerberth, B., Lee, J.-Y., Bergman, T., Carlquist, M., Boman, H. G., Mutt, V., and Jornvall, H. (1991) Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. Eur. J. Biochem. 202, 849–854.PubMedCrossRefGoogle Scholar
  25. 25.
    Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S. S. L., Daher, K., Bainton, D. F., and Lehrer, R. I. (1985) Defensins. Natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76, 1427–1435.PubMedCrossRefGoogle Scholar
  26. 26.
    Guo, D., Mant, C. T., and Hodges, R. S. (1987) Effects of ion-pairing reagents on the prediction of peptide retention in re versed-phase high performance liquid chromatography. J. Chrom. 386, 205–222.CrossRefGoogle Scholar
  27. 27.
    Selsted, M. E. and Becker, H. W., III (1986) Eosin Y a reversible stain for detecting electrophoretically resolved protein. Anal. Biochem. 155, 270–274.PubMedCrossRefGoogle Scholar
  28. 28.
    Anderson, J. K. and Mole, J. E. (1983) Adaptation of reverse-phase high-performance liquid chromatography for the isolation and sequence analysis of peptides from plasma amyloid p-component, in High-Performance Liquid Chromatography of Proteins and Peptides (Hearn, M. T. W., Regnier, F. E., and Wehr, C. T., eds.), Academic, New York, pp. 29–37.Google Scholar
  29. 29.
    Selsted, M. E. (1993) Investigational approaches for studying the structures and biological functions of myeloid antimicrobial peptides, in Genetic Engineering: Principles and Methods (Setlow, J. K., ed.), Plenum, New York, pp 131–147.Google Scholar
  30. 30.
    Bennett, H. P. J., Browne, C. A., and Solomon, S (1983) α-N-Acetyl-β-Endorphin1-26 from the neurointermediary lobe of the rat pituitary isolation, purification, and characterization by high-performance liquid chromatography, in High-Performance Liquid Chromatography of Proteins and Peptides (Hearn, M. T. W., Regnier, F. E., and Wehr, C. T., eds.), Academic, New York, p 253–261.Google Scholar
  31. 31.
    Eisenhauer, P. B., Harwig, S. S. L., Szklarek, D., Ganz, T., Selsted, M. E., and Lehrer, R. I. (1989) Purification and antimicrobial properties of three defensins from rat neutrophils. Infect. Immun. 57, 2021–2027.PubMedGoogle Scholar
  32. 32.
    Selsted, M. E., Brown, D. M., DeLange, R. J., and Lehrer, R. I. (1983) Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages. J. Biol. Chem. 258, 14,485–14,489.PubMedGoogle Scholar
  33. 33.
    Harwig, S. S. L., Swiderek, K. M., Kokryakov, V. N., Tan, L., Lee, T. D., Panyutich, E. A., Aleshina, G. M., Shamova, O. V., and Lehrer, R. I. (1994) Gallinacins cysteine-rich antimicrobial peptides of chicken leukocytes. FEBS Lett. 342, 281–285.PubMedCrossRefGoogle Scholar
  34. 34.
    Romeo, D., Skerlavaj, B., Bolognesi, M., and Gennaro, R. (1988) Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils. J. Biol. Chem. 263, 9573–9575.PubMedGoogle Scholar
  35. 35.
    Hultmark, D., Steiner, H., Rasmuson, T., and Boman, H. G. (1980) Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur. J. Biochem. 106, 7–16.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Michael E. Selsted
    • 1
  1. 1.Department of Pathology, College of MedicineUniversity of CaliforniaIrvine

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