Methods for Assessments of Collagenolytic Activity of the Vibrio cholerae Extracellular Proteases, Purification of Secreted Collagenase VchC, and Extraction of Type I Collagen from Fish Skin

  • Bo R. Weber
  • Aleksandra E. SikoraEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1839)


Secreted proteases have been linked to facilitating the survival of Vibrio cholerae in different environmental niches. Examination of protease activity using various measures is critical to determine the substrate specificity of a given proteolytic enzyme and its biological function. Here, we describe methods to assess collagenolytic activity of the V. cholerae extracellular proteases using synthetic and natural substrates in quantitative and qualitative assays. Purification of the secreted V. cholerae collagenase VchC and extraction of type I collagen from fish skin are also described.

Key words

Metalloprotease Collagenolytic proteases Secretion Collagenase Type I collagen SDS-PAGE FALGPA Zymography Affinity chromatography DQ-gelatin 



This work was supported by the start-up funds from Oregon State University (OSU) as well as Faculty Development Funding to AES.


  1. 1.
    Watanabe K (2004) Collagenolytic proteases from bacteria. Appl Microbiol Biotechnol 63(5):520–526. CrossRefPubMedGoogle Scholar
  2. 2.
    Harrington DJ (1996) Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun 64(6):1885–1891PubMedPubMedCentralGoogle Scholar
  3. 3.
    Duarte AS, Correia A, Esteves AC (2014) Bacterial collagenases—a review. Crit Rev Microbiol.
  4. 4.
    Park BR, Zielke RA, Wierzbicki IH, Mitchell KC, Withey JH, Sikora AE (2015) A metalloprotease secreted by the type II secretion system links Vibrio cholerae with collagen. J Bacteriol 197(6):1051–1064. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Halpern M, Gancz H, Broza M, Kashi Y (2003) Vibrio cholerae hemagglutinin/protease degrades chironomid egg masses. Appl Environ Microbiol 69(7):4200–4204CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Vaitkevicius K, Rompikuntal PK, Lindmark B, Vaitkevicius R, Song T, Wai SN (2008) The metalloprotease PrtV from Vibrio cholerae. FEBS J 275(12):3167–3177. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10(3):211–220. CrossRefPubMedGoogle Scholar
  8. 8.
    Van Wart HE, Steinbrink DR (1981) A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Anal Biochem 113(2):356–365CrossRefPubMedGoogle Scholar
  9. 9.
    Wingfield P (2001) Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci. Appendix 3:Appendix 3F.
  10. 10.
    Miller EJ, Gay S (1987) The collagens: an overview and update. Methods Enzymol 144:3–41CrossRefPubMedGoogle Scholar
  11. 11.
    Exposito JY, Valcourt U, Cluzel C, Lethias C (2010) The fibrillar collagen family. Int J Mol Sci 11(2):407–426. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Suphatharaprateep W, Cheirsilp B, Jongjareonrak A (2011) Production and properties of two collagenases from bacteria and their application for collagen extraction. New Biotechnol 28(6):649–655. CrossRefGoogle Scholar
  13. 13.
    Zielke RA, Simmons RS, Park BR, Nonogaki M, Emerson S, Sikora AE (2014) The type II secretion pathway in Vibrio cholerae is characterized by growth phase-dependent expression of exoprotein genes and is positively regulated by sigmaE. Infect Immun 82(7):2788–2801. CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, College of PharmacyOregon State UniversityCorvallisUSA

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