High Level Activity of Recombinant Lysostaphin After Computer Simulation and Additive-Based Refolding

  • Shabnam Sadoogh Abbasian
  • Safieh Soufian
  • Ehsanollah Ghaznavi-Rad
  • Hamid AbtahiEmail author


Lysostaphin is a peptidoglycan hydrolase, produced by Staphylococcus simulans, which has illustrated significant bactericidal activities against Staphylococcus aureus species. Currently, recombination is the common approach of lysostaphin production. However, the recombinant produced lysostaphin shows weak antibacterial activities. The reason can be the aggregation of produced lysostaphin which leads to the destruction of natural protein folding. The most common strategy providing the best situation for correct refolding of the recombinant protein is dialysis. In this study, based on the computer simulations different condition of dialysis was applied to achieve the most significant antibacterial activity. In this study, lysostaphin was expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS cells and purified by affinity chromatography. Various dialysis methods were employed to enable the protein to refold to its natural form. The results of final protein antibacterial activity evidenced the high efficiency of computer simulations estimation ability in predicting best dialysis buffer according to the interaction between protein and buffer additive compounds. Finally, it was confirmed that the buffer containing proline 0.15 M and glucose 0.2 M caused the best lysostaphin refolding. Employing computer simulation before initiating the dialysis process would be a novel efficient and economic pathway of protein folding recovery.


Computer simulation Lysostaphin Protein refolding Recombinant protein Staphylococcus aureus 



This Study was conducted with financial assistance from Arak University of Medical Sciences, Iran, and the authors are grateful for the university’s invaluable contribution to this study.


This study was emanated from a proposal (No: 1379) approved and supported by Arak University of Medical Sciences, Iran. This study was approved by the ethics committee of Arak University of Medical Sciences, Arak, Iran, Ethics code: 91-128-6.

Compliance with Ethical Standards

Conflict of interest

The authors declare that no conflict of interest exists.


  1. Abbasian SS, Ghaznavi Rad E, Akbari N, Zolfaghari MR, Pakzad I, Abtahi H (2015) Overexpression and enzymatic assessment of antigenic fragments of hyaluronidase recombinant protein from Streptococcus pyogenes. Jundishapur J Microbiol 8(1):e13653-e13653Google Scholar
  2. Aguzzi A, O’connor T (2010) Protein aggregation diseases: pathogenicity and therapeutic perspectives. Nat Rev Drug Discov 9(3):237CrossRefGoogle Scholar
  3. Akbari N, Khajeh K, Ghaemi N, Salemi Z (2010) Efficient refolding of recombinant lipase from Escherichia coli inclusion bodies by response surface methodology. Protein Expr Purif 70(2):254–259PubMedCrossRefGoogle Scholar
  4. Bai Y, Watt B, Wahome PG, Mantis NJ, Robertus JD (2010) Identification of new classes of ricin toxin inhibitors by virtual screening. Toxicon 56(4):526–534PubMedPubMedCentralCrossRefGoogle Scholar
  5. Bastos MdCdF, Coutinho BG, Coelho MLV (2010) Lysostaphin: a staphylococcal bacteriolysin with potential clinical applications. Pharmaceuticals 3(4):1139–1161PubMedPubMedCentralCrossRefGoogle Scholar
  6. Caffery ML, Dobosh PA, Richardson D (1998) Laboratory exercises using HyperChem. Hypercube, GainesvilleGoogle Scholar
  7. Conchillo-Solé O, de Groot NS, Avilés FX, Vendrell J, Daura X, Ventura S (2007) AGGRESCAN: a server for the prediction and evaluation of” hot spots” of aggregation in polypeptides. BMC Bioinf 8(1):65CrossRefGoogle Scholar
  8. Dajcs JJ, Hume EB, Moreau JM, Caballero AR, Cannon BM, O’Callaghan RJ (2000) Lysostaphin treatment of methicillin-resistant Staphylococcus aureus keratitis in the rabbit. Investig Ophthalmol Vis Sci 41(6):1432–1437Google Scholar
  9. Farhangnia L, Ghaznavi-Rad E, Mollaee N, Abtahi H (2014) Cloning, expression, and purification of recombinant Lysostaphin from Staphylococcus simulans. Jundishapur J Microbiol 7(5):e10009PubMedPubMedCentralCrossRefGoogle Scholar
  10. Farjadi V, Abtahi H, Zolfaghari MR, Soufian S, Hasanzadeh L (2013) Expression, purification and evaluation of antigenicity of caga antigenic fragment of helicobacter pylori. Jundishapur J Microbiol 6(9):1–6CrossRefGoogle Scholar
  11. Gründling A, Schneewind O (2006) Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus. J Bacteriol 188(7):2463–2472PubMedPubMedCentralCrossRefGoogle Scholar
  12. Hamada H, Arakawa T, Shiraki K (2009) Effect of additives on protein aggregation. Curr Pharm Biotechnol 10(4):400–407PubMedCrossRefGoogle Scholar
  13. King BF, Biel ML, Wilkinson BJ (1980) Facile penetration of the Staphylococcus aureus capsule by lysostaphin. Infect Immun 29(3):892–896PubMedPubMedCentralGoogle Scholar
  14. Kumat T, Samuel D, Jayaraman G, Srimathi T, Yu C (1998) The role of proline in the prevention of aggregation during protein folding in vitro. IUBMB Life 46(3):509–517CrossRefGoogle Scholar
  15. Leibly DJ, Nguyen TN, Kao LT, Hewitt SN, Barrett LK, Van Voorhis WC (2012) Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins. PLoS ONE 7(12):e52482PubMedPubMedCentralCrossRefGoogle Scholar
  16. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339(8):520–532. PubMedCrossRefGoogle Scholar
  17. Macchi F, Eisenkolb M, Kiefer H, Otzen DE (2012) The effect of osmolytes on protein fibrillation. Int J Mol Sci 13(3):3801–3819PubMedPubMedCentralCrossRefGoogle Scholar
  18. Molaee N, Abtahi H, Mosayebi G (2013) Expression of recombinant streptokinase from Streptococcus pyogenes and its reaction with infected human and murine sera. Iran J Basic Med Sci 16(9):985PubMedPubMedCentralGoogle Scholar
  19. Mondal S, Shet D, Prasanna C, Atreya HS (2013) High yield expression of proteins in E. coli for NMR studies. Adv Biosci Biotechnol 4(06):751CrossRefGoogle Scholar
  20. Nuc P, Nuc K (2006) Recombinant protein production in Escherichia coli. Postepy Biochem 52(4):448–456PubMedGoogle Scholar
  21. Palmer I, Wingfield PT (2004) Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protocols Protein Sci 6.3(1–6.3):20Google Scholar
  22. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612CrossRefGoogle Scholar
  23. Samuel D, Kumar TKS, Ganesh G, Jayaraman G, Yang P-W, Chang M-M, Chang D-K (2000) Proline inhibits aggregation during protein refolding. Protein Sci 9(2):344–352PubMedPubMedCentralCrossRefGoogle Scholar
  24. Satishkumar R, Sankar S, Yurko Y, Lincourt A, Shipp J, Heniford BT, Vertegel A (2011) Evaluation of the antimicrobial activity of lysostaphin-coated hernia repair meshes. Antimicrob Agents Chemother 55(9):4379–4385PubMedPubMedCentralCrossRefGoogle Scholar
  25. Schindler CA, Schuhardt VT (1965) Purification and properties of lysostaphin—a lytic agent for Staphylococcus aureus. Biochim Biophys Acta (BBA) 97(2):242–250CrossRefGoogle Scholar
  26. Sheagren JN (1984) Staphylococcus aureus: the persistent pathogen. N Engl J Med 310(22):1437–1442PubMedCrossRefGoogle Scholar
  27. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99(4):303–310PubMedCrossRefGoogle Scholar
  28. Swartz JR (2001) Advances in Escherichia coli production of therapeutic proteins. Curr Opin Biotechnol 12(2):195–201PubMedCrossRefGoogle Scholar
  29. Vagenende V, Yap MG, Trout BL (2009) Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry 48(46):11084–11096PubMedCrossRefGoogle Scholar
  30. Waitz JA (1990) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, VillanovaGoogle Scholar
  31. Wallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein engineering design selection 8(2):127–134CrossRefGoogle Scholar
  32. Williamson CM, Bramley A, Lax A (1994) Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system. Appl Environ Microbiol 60(3):771–776PubMedPubMedCentralGoogle Scholar
  33. Wingfield PT (2015) Overview of the purification of recombinant proteins. Curr Protoc Protein Sci. PubMedPubMedCentralCrossRefGoogle Scholar
  34. Wu J, Wang J, Li M, Yang Y, Wang B, Zheng YG (2011) Small molecule inhibitors of histone acetyltransferase Tip60. Bioorg Chem 39(1):53–58PubMedCrossRefGoogle Scholar
  35. Yamaguchi S, Yamamoto E, Mannen T, Nagamune T (2013) Protein refolding using chemical refolding additives. Biotechnol J 8(1):17–31PubMedCrossRefGoogle Scholar
  36. Zhang T, Xu X, Shen L, Feng Y, Yang Z, Shen Y, Wang X (2009) Modeling of protein refolding from inclusion bodies. Acta Biochim Biophys Sin 41(12):1044–1052PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Shabnam Sadoogh Abbasian
    • 1
  • Safieh Soufian
    • 2
  • Ehsanollah Ghaznavi-Rad
    • 1
  • Hamid Abtahi
    • 1
    Email author
  1. 1.Molecular and Medicine Research Center, School of MedicineArak University of Medical SciencesArakIran
  2. 2.Payame Noor UniversityTehranIran

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