Advertisement

Biochemistry (Moscow)

, Volume 81, Issue 5, pp 502–510 | Cite as

Staphylococcus simulans recombinant lysostaphin: Production, purification, and determination of antistaphylococcal activity

  • I. S. BokshaEmail author
  • N. V. Lavrova
  • A. V. Grishin
  • A. V. Demidenko
  • A. M. Lyashchuk
  • Z. M. Galushkina
  • R. S. Ovchinnikov
  • A. M. Umyarov
  • L. R. Avetisian
  • M. Iu. Chernukha
  • I. A. Shaginian
  • V. G. Lunin
  • A. S. Karyagina
Article

Abstract

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

Keywords

Staphylococcus simulans Escherichia coli recombinant lysostaphin heterologous expression liquid chromatography lytic activity Staphylococcus sp. clinical isolates 

Abbreviations

IPTG

isopropyl-1-thio-ß-D-galactopyra-noside

PIZ

partial inhibition zone

PMSF

phenylmethylsul-fonyl fluoride

TIZ

total inhibition zone

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bastos, M. C. F., Coutinho, B. G., and Coelho, M. L. V. (2010) Lysostaphin: a staphylococcal bacteriolysin with potential clinical applications, Pharmaceuticals, 3, 11391161.CrossRefGoogle Scholar
  2. 2.
    Szweda, P., Schielmann, M., Kotlowski, R., Gorczyca, G., Zalewska, M., and Milewski, S. (2012) Peptidoglycan hydrolases–potential weapons against Staphylococcus aureus, Appl. Microbiol. Biotechnol., 96, 1157–1174.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Szweda, P., Gorczyca, G., Filipkowski, P., Zalewska, M., and Milewski, S. (2014) Efficient production of Staphylococcus simulans lysostaphin in a benchtop bioreactor by recombinant Escherichia coli, Prep. Biochem. Biotechnol., 44, 370–381.CrossRefPubMedGoogle Scholar
  4. 4.
    Zhao, H., Blazanovic, K., Choi, Y., Bailey-Kellogg, C., and Griswold, K. E. (2014) Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris, Appl. Environ. Microbiol., 80, 2746–2753.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kumar, J. K. (2008) Lysostaphin: an antistaphylococcal agent, Appl. Microbiol. Biotechnol., 80, 555–561.CrossRefPubMedGoogle Scholar
  6. 6.
    Kumar, A., Khan, I. A., Sharma, P. R., Sumathy, K., and Krishna, M. E. (2014) Evaluation of activity of recombinant lysostaphin against isolates of meticillin-resistant Staphylococcus aureus from Indian hospitals, J. Med. Microbiol., 63, 763–766.CrossRefPubMedGoogle Scholar
  7. 7.
    Chan, E.-C. (1996) Expression and purification of recombinant lysostaphin in Escherichia coli, Biotechnol. Lett., 18, 833–838.CrossRefGoogle Scholar
  8. 8.
    Szweda, P., Pladzyk, R., Kotlowski, R., and Kur, J. (2001) Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein–chitin-binding domain (CBD) system, Protein Express. Purif., 22, 467–471.CrossRefGoogle Scholar
  9. 9.
    Szweda, P., Kotowski, R., and Kur, J. (2005) Protective effect of lysostaphin from Staphylococcus simulans against growth of Staphylococcus aureus in milk and some other food products, J. Biotechnol., 117, 203–213.CrossRefPubMedGoogle Scholar
  10. 10.
    Farhangnia, L., Ghaznavi-Rad, E., Mollaee, N., and Abtahi, H. (2014) Cloning, expression, and purification of recombinant lysostaphin from Staphylococcus simulans, Jundishapur J. Microbiol., 7, e10009.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sharma, R., Sharma, P. R., Choudhary, M. L., Pande, A., and Khatri, G. S. (2006) Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli, Protein Express. Purif., 45, 206–215.CrossRefGoogle Scholar
  12. 12.
    Szweda, P., Gorczyca, G., Tylingo, R., Kurlenda, J., Kwiecinski, J., and Milewski, S. (2014) Chitosan–protein scaffolds loaded with lysostaphin as potential antistaphylococcal wound dressing materials, J. Appl. Microbiol., 117, 634–642.CrossRefPubMedGoogle Scholar
  13. 13.
    Recsei, P. A. (1990) Expression of recombinant mature lysostaphin, US Patent 4931390.Google Scholar
  14. 14.
    Williamson, C. M., Bramley, A. J., and Lax, A. J. (1994) Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system, Appl. Environ. Microbiol., 60, 771–776.PubMedPubMedCentralGoogle Scholar
  15. 15.
    Bardelang, P., Vankemmelbeke, M., Zhang, Y., Jarvis, H., Antoniadou, E., Rochette, S., Thomas, N. R., Penfold, C. N., and James, R. (2009) Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin, Biochem. J., 418, 615–624.CrossRefPubMedGoogle Scholar
  16. 16.
    Warfield, R., Bardelang, P., Saunders, H., Chan, W. C., Penfold, C., James, R., and Thomas, N. R. (2006) Internally quenched peptides for the study of lysostaphin: an antimicrobial protease that kills Staphylococcus aureus, Org. Biomol. Chem., 4, 3626–3638.CrossRefPubMedGoogle Scholar
  17. 17.
    Khatri, G. S., and Sharma, R. (2009) Expression of recombinant mature lysostaphin, Patent EP1224271 B1.Google Scholar
  18. 18.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.Google Scholar
  19. 19.
    Schindler, C. A., and Schuardt, V. T. (1965) Purification and properties of lysostaphin: a lytic agent for the Staphylococcus aureus, Biochim. Biophys. Acta, 97, 242250.Google Scholar
  20. 20.
    Schindler, C. A. (1965) The role of NaCl in the lysis of Staphylococcus aureus by lysostaphin, J. Gen. Microbiol., 40, 199–205.CrossRefPubMedGoogle Scholar
  21. 21.
    Marova, I., and Kovar, J. (1993) Spectrophotometric detection of bacteriolytic activity of diluted lysostaphin solutions, Folia Microbiol., 38, 153–158.CrossRefGoogle Scholar
  22. 22.
    Huang, Q., Lu, H., and Lu, W. (2009) Method of Secretory Expression of Lysostaphin in Escherichia coli at High Level, Patent WO 20090186380.Google Scholar
  23. 23.
    Zhou, R., Chen, S., and Recsei, P. (1988) A dye release assay for determination of lysostaphin activity, Anal. Biochem., 171, 141–144.CrossRefPubMedGoogle Scholar
  24. 24.
    Sabala, I., Jonsson, I., Tarkowski, A., and Bochtler, M. (2012) Anti-staphylococcal activities of lysostaphin and LytM catalytic domain, BMC Microbiol., 12, 97.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wu, J. A., Kusuma, C., Mond, J. J., and Kokai-Kun, J. F. (2003) Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces, Antimicrob. Agents Chemother., 47, 3407–3414.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Yang, X.-Y., Li, C.-R., Lou, R.-H., Wang, Y.-M., Zhang, W.-X., Chen, H.-Z., Huang, Q.-S., Han, Y.-X., Jiang, J.D., and You, X.-F. (2007) In vitro activity of recombinant lysostaphin against Staphylococcus aureus isolates from hospitals in Beijing, China, J. Med. Microbiol., 56, 71–76.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2016

Authors and Affiliations

  • I. S. Boksha
    • 1
    Email author
  • N. V. Lavrova
    • 1
  • A. V. Grishin
    • 1
  • A. V. Demidenko
    • 1
  • A. M. Lyashchuk
    • 1
  • Z. M. Galushkina
    • 1
  • R. S. Ovchinnikov
    • 1
  • A. M. Umyarov
    • 1
  • L. R. Avetisian
    • 1
  • M. Iu. Chernukha
    • 1
  • I. A. Shaginian
    • 1
  • V. G. Lunin
    • 1
  • A. S. Karyagina
    • 1
  1. 1.N. F. Gamaleya Federal Research Centre for Epidemiology and MicrobiologyMinistry of Health of the Russian FederationMoscowRussia

Personalised recommendations