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.
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Abbreviations
- IPTG:
-
isopropyl-1-thio-ß-D-galactopyra-noside
- PIZ:
-
partial inhibition zone
- PMSF:
-
phenylmethylsul-fonyl fluoride
- TIZ:
-
total inhibition zone
References
Bastos, M. C. F., Coutinho, B. G., and Coelho, M. L. V. (2010) Lysostaphin: a staphylococcal bacteriolysin with potential clinical applications, Pharmaceuticals, 3, 11391161.
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.
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.
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.
Kumar, J. K. (2008) Lysostaphin: an antistaphylococcal agent, Appl. Microbiol. Biotechnol., 80, 555–561.
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.
Chan, E.-C. (1996) Expression and purification of recombinant lysostaphin in Escherichia coli, Biotechnol. Lett., 18, 833–838.
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.
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.
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.
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.
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.
Recsei, P. A. (1990) Expression of recombinant mature lysostaphin, US Patent 4931390.
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.
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.
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.
Khatri, G. S., and Sharma, R. (2009) Expression of recombinant mature lysostaphin, Patent EP1224271 B1.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
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.
Schindler, C. A. (1965) The role of NaCl in the lysis of Staphylococcus aureus by lysostaphin, J. Gen. Microbiol., 40, 199–205.
Marova, I., and Kovar, J. (1993) Spectrophotometric detection of bacteriolytic activity of diluted lysostaphin solutions, Folia Microbiol., 38, 153–158.
Huang, Q., Lu, H., and Lu, W. (2009) Method of Secretory Expression of Lysostaphin in Escherichia coli at High Level, Patent WO 20090186380.
Zhou, R., Chen, S., and Recsei, P. (1988) A dye release assay for determination of lysostaphin activity, Anal. Biochem., 171, 141–144.
Sabala, I., Jonsson, I., Tarkowski, A., and Bochtler, M. (2012) Anti-staphylococcal activities of lysostaphin and LytM catalytic domain, BMC Microbiol., 12, 97.
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.
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.
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Published in Russian in Biokhimiya, 2016, Vol. 81, No. 5, pp. 668-677.
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Boksha, I.S., Lavrova, N.V., Grishin, A.V. et al. Staphylococcus simulans recombinant lysostaphin: Production, purification, and determination of antistaphylococcal activity. Biochemistry Moscow 81, 502–510 (2016). https://doi.org/10.1134/S0006297916050072
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DOI: https://doi.org/10.1134/S0006297916050072