Acta Biologica Hungarica

, Volume 59, Issue 3, pp 289–304 | Cite as

Purification and Characterization of a Thermal Stable Antimicrobial Protein From Housefly Larvae. Musca Domestica, Induced by Ultrasonic Wave

  • Y. J. HaoEmail author
  • Y. J. Jing
  • H. Qu
  • D. S. Li
  • R. Q. Du


This work describes the induction, purification and partial biochemical characterizations of an antimicrobial protein from the housefly larvae induced by ultrasonic wave. It has been purified to apparent homogeneity by ammonium sulfate precipitation followed by Sephadex G-75, Bio-gel P6 gel filtration, and CM-Sepharose Fast Flow cation exchange chromatography. The protein is a cationic protein with an apparent molecular weight of 16315 Da determined by no-denaturing electrophoresis and SDS-PAGE, respectively. Biochemical profile assays show that this protein has good thermal stability, and repeatedly frozen and defrosted durability. The optimum pH for antimicrobial activity is around pH5. The antimicrobial range of the protein includes Gram-positive, Gram-negative bacteria and some fungi. Results of the membrane permeability assays suggest that the probable mode of action of this protein is membrane-disrupting mechanism.


Housefly Musca domestica antimicrobial protein antimicrobial activity membrane permeability 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Akuffo, H., Hultmark, D., Engstom, A., Frohlich, D., Kimbrell, D. (1998) Drosophila antibacterial protein, cecropin A, differentially affects non-bacterial organisms such a. Leishmania in a manner different from other amphipathic peptides. Int. J. Mol. Med. 1, 77–82.PubMedGoogle Scholar
  2. 2.
    An, C., Li, D., Du, R. (2004) Analysis of antibacterial-relative proteins and peptides in housefly larvae. Wei Sheng Yan Jiu 33, 86–88.PubMedGoogle Scholar
  3. 3.
    Baumann, G., Mueller, P. (1974) A molecular model of membrane excitability. J. Supramol. Struct. 2, 538–557.CrossRefGoogle Scholar
  4. 4.
    Boheim, G. (1974) Statistical analysis of alamethicin channels in black lipid membranes. J. Membr. Biol. 19, 277–303.CrossRefGoogle Scholar
  5. 5.
    Boman, H. G. (1991) Antibacterial peptides: key components needed in immunity. Cell 65, 205–207.CrossRefGoogle Scholar
  6. 6.
    Boman, H. G. (1996) Peptide antibiotics: holy or heretic grails of innate immunity. Scand. J. Immunol. 43, 475–482.CrossRefGoogle Scholar
  7. 7.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.CrossRefGoogle Scholar
  8. 8.
    Brey, P. T., Lee, W. I., Yamakawa, M., Koizumi, Y., Perrot, S., Francois, M., Ashida, M. (1993) Role of the integument in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. Proc. Natl. Acad. Sci. USA, 90, 6275–6279.CrossRefGoogle Scholar
  9. 9.
    Bulet, P., Stocklin, R., Menin, L. (2004) Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169–184.CrossRefGoogle Scholar
  10. 10.
    Carlsson, A., Engstrom, P., Palva, E. T., Bennich, H. (1991) Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins i. Escherichia coli by interfering with omp gene transcription. Infect Immun. 59, 3040–3045.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Carson, C. F., Hammer, K. A., Riley, T. V. (1995) Broth micro-dilution method for determining the susceptibility o. Escherichia coli an. Staphylococcus aureus to the essential oi. of Melaleuca alterni-folia (tea tree oil). Microbios. 82, 181–185.PubMedGoogle Scholar
  12. 12.
    Casteels, P. (1990) Possible applications of insect antibacterial peptides. Res. Immunol. 141, 940–942.CrossRefGoogle Scholar
  13. 13.
    Daffre, S., Kylsten, P., Samakovlis, C., Hultmark, D. (1994) The lysozyme locus i. Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract. Mol. Gen. Genet. 242, 152–162.CrossRefGoogle Scholar
  14. 14.
    Engstrom, P., Carlsson, A., Engstrom, A., Tao, Z. J., Bennich, H. (1984) The antibacterial effect of attacins from the silk mot. Hyalophora cecropia is directed against the outer membrane o. Escherichia coli. Embo. J. 3, 3347–3351.CrossRefGoogle Scholar
  15. 15.
    Fujiwara, S., Imai, J., Fujiwara, M, Yaeshima, T., Kawashima, T., Kobayashi, K. (1990) A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royal-isin. J. Biol. Chem. 265, 11333–11337.PubMedGoogle Scholar
  16. 16.
    Geng, H., An, C. J., Hao, Y. J., Li, D. S., Du, R. Q. (2004) Molecular cloning and expression of attacin from housefl. (Musca domestica). Yi Chuan Xue Bao 31, 1344–1350.PubMedGoogle Scholar
  17. 17.
    Guo, Y. M., Dai, Z. Y., Hu, Y. L. (1995) Study on the properties and anticancer activities of antibacterial protein. J. Nanjing University (Natural Science), 62–67.Google Scholar
  18. 18.
    Hancock, R. E. (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis. 1, 156–164.CrossRefGoogle Scholar
  19. 19.
    Hetru, C., Bulet, P. (1997) Strategies for the isolation and characterization of antimicrobial peptides of invertebrates. Methods Mol. Biol. 78, 35–49.PubMedGoogle Scholar
  20. 20.
    Hetru, C., Hoffmann, D., Bulet, P., Brey, P., Hultmark, D. (1998) Molecular Mechanisms of Immune Responses in Insects. Chapman and Hall, New York, pp. 40–66.Google Scholar
  21. 21.
    Hou, L., Shi, Y., Zhai, P., Le, G. (2007) Antibacterial activity and in vitro anti-tumor activity of the extract of the larvae of the housefl. (Musca domestica). J. Ethnopharmacol. 111, 227–231.CrossRefGoogle Scholar
  22. 22.
    Huang, H. W. (2000) Action of antimicrobial peptides: two-state model. Biochemistry 39, 8347–8352.CrossRefGoogle Scholar
  23. 23.
    Hultmark, D., Engstrom, A., Bennich, H., Kapur, R., Boman, H. G. (1982) Insect immunity: isolation and structure of cecropin D and four minor antibacterial components fro. Cecropia pupae. Eur. J. Biochem. 127, 207–217.CrossRefGoogle Scholar
  24. 24.
    Jin, F., Xu, X., Zhang, W., Gu, D. (2006) Expression and characterization of a housefly cecropin gene in the methylotrophic yeast. Pichiapastoris. Protein Expr. Purif 49, 39–46.CrossRefGoogle Scholar
  25. 25.
    Jin, H. Y., Hu, Q., Jun, J. Y., Ju, A., Sen, L. D., Qian, D. R., Lin, Q. R. (2005) Preliminary studies on the zinc-induced metallothionein protein with antibacterial activity in housefly larvae. Musca domestica. Acta Biol. Hung. 56, 283–295.CrossRefGoogle Scholar
  26. 26.
    Jolies, J., Schoentgen, F., Croizier, G., Croizier, L., Jolies, P. (1979) Insect lysozymes from three species of Lepidoptera: their structural relatedness to the C (chicken) type lysozyme. J. Mol. Evol. 14, 267–271.CrossRefGoogle Scholar
  27. 27.
    Kylsten, P., Kimbrell, D. A., Daffre, S., Samakovlis, C., Hultmark, D. (1992) The lysozyme locus in Drosophila melanogaster: different genes are expressed in midgut and salivary glands. Mol. Gen. Genet. 232, 335–343.CrossRefGoogle Scholar
  28. 28.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle Scholar
  29. 29.
    Lehrer, R. I., Barton, A., Ganz, T. (1988) Concurrent assessment of inner and outer membrane per-meabilization and bacteriolysis i. E. coli by multiple-wavelength spectrophotometry. J. Immunol. Methods 108, 153–158.CrossRefGoogle Scholar
  30. 30.
    Liang, Y., Wang, J. X., Zhao, X. F., Du, X. J., Xue, J. F. (2006) Molecular cloning and characterization of cecropin from the housefl. (Musca domestica), and its expression i. Escherichia coli. Dev. Comp. Immunol. 30, 249–257.CrossRefGoogle Scholar
  31. 31.
    Martinez, M. B., Flickinger, M. C., Nelsestuen, G. L. (1996) Accurate kinetic modeling of alkaline phosphatase in th. Escherichia coli periplasm: implications for enzyme properties and substrate diffusion. Biochemistry 35, 1179–1186.CrossRefGoogle Scholar
  32. 32.
    Reisfeld, R. A., Lewis, U. J., Brink, N. G., Steelman, S. L. (1962) Human growth hormone: preparation from acetone powder. Endocrinology 71, 559–563.CrossRefGoogle Scholar
  33. 33.
    Rosetto, M., Manetti, A. G., Giordano, P. C., Marri, L., Amons, R., Baldari, C. T., Marchini, D., Dallai, R. (1996) Molecular characterization of ceratotoxin C., a novel antibacterial female-specific peptide of the ceratotoxin family from the medfl. Ceratitis capitata. Eur. J. Biochem. 241, 330–337.CrossRefGoogle Scholar
  34. 34.
    Samakovlis, C., Kylsten, P., Kimbrell, D. A., Engstrom, A., Hultmark, D. (1991) The andropin gene and its product, a male-specific antibacterial peptide i. Drosophila melanogaster. Embo J. 10, 163–169.CrossRefGoogle Scholar
  35. 35.
    Shai, Y. (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462, 55–70.CrossRefGoogle Scholar
  36. 36.
    Silvestro, L., Weiser, J. N., Axelsen, P. H. (2000) Antibacterial and antimembrane activities of cecropin A i. Escherichia coli. Antimicrob. Agents Chemother. 44, 602–607.CrossRefGoogle Scholar
  37. 37.
    Sondergaard, L. (1993) Homology between the mammalian liver and th. Drosophila fat body. Trends Genet. 9, 193.CrossRefGoogle Scholar

Copyright information

© Akadémiai Kiadó, Budapest 2008

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Y. J. Hao
    • 1
    • 3
    Email author
  • Y. J. Jing
    • 2
  • H. Qu
    • 1
  • D. S. Li
    • 1
  • R. Q. Du
    • 1
  1. 1.Department of Cell and Genetics, Genetic Engineering LaboratoryCollege of Life Science, Nankai UniversityTian JinChina
  2. 2.Department of Bioengineering, College of Chemical EngineeringHebei University of TechnologyTian JinChina
  3. 3.Departamento de BiologiaUniversidade dos AcoresPonta Delgada, AzoresPortugal

Personalised recommendations