Skip to main content
SpringerLink
Log in

HPLC Methods for Purification of Antimicrobial Peptides

  • Protocol
Antibacterial Peptide Protocols

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 78))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 62–92.

    Article  Google Scholar 

  2. Martin, E., Ganz, T., and Lehrer, R. I. (1995) Defensins and other endogenous peptide antibiotics of vertebrates. J. Leukocyte Biol. 58, 128–136.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  10. Schonwetter, B. S., Stolzenberg, E. D., and Zasloff, M. A. (1995) Epithelial antibiotics induced at sites of inflammation. Science 267, 1645–1648.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  13. Bevins, C. L. and Zasloff, M. (1990) Peptides from frog skin. Ann. Rev. Biochem. 59, 395–414.

    Article  PubMed  CAS  Google Scholar 

  14. Hoffmann, J. A. and Hetru, C. (1992) Insect defensins, inducible antibacterial peptides. Immunol. Today 13, 411–415.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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. 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. 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. 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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, College of Medicine, University of California, Irvine, CA

    Michael E. Selsted

Authors
  1. Michael E. Selsted
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Emory University School of Medicine, Atlanta, GA

    William M. Shafer

Rights and permissions

Reprints and permissions

Copyright information

© 1997 Humana Press Inc.

About this protocol

Cite this protocol

Selsted, M.E. (1997). HPLC Methods for Purification of Antimicrobial Peptides. In: Shafer, W.M. (eds) Antibacterial Peptide Protocols. Methods In Molecular Biology™, vol 78. Humana Press. https://doi.org/10.1385/0-89603-408-9:17

Download citation

  • DOI: https://doi.org/10.1385/0-89603-408-9:17

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-408-2

  • Online ISBN: 978-1-59259-564-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation