Advertisement

Journal of Industrial Microbiology & Biotechnology

, Volume 42, Issue 10, pp 1369–1376 | Cite as

Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector

  • Licong Zhang
  • Xiaodan Li
  • Dandan Wei
  • Jue Wang
  • Anshan ShanEmail author
  • Zhongyu Li
Biotechnology Methods
First Online:

Abstract

Plectasin, the first fungus defensin, is especially efficient against Gram-positive bacteria. To explore an effective approach for expressing plectasin in Bacillus subtilis, the sequence encoding plectasin fused with the small ubiquitin-like modifier (SUMO) gene, the 6 × His gene and the signal peptide of SacB were cloned into an E. coli–B. subtilis shuttle vector pGJ148 in which the maltose utilization operon promoter Pglv directed the expression. The fusion protein successfully secreted in culture and approximately, 41 mg of the recombinant fusion protein SUMO-plectasin was purified per liter of culture supernatant. After purification by Ni-NTA resin column and digestion by SUMO protease, 5.5 mg of plectasin with a purity of 94 % was obtained from 1 L fermentation culture. Recombinant plectasin was found inhibition activity against S. pneumoniae, S. aureus and S. epidermidis. These results indicate that the maltose-induced expression system may be a safe and efficient way for the large-scale production of soluble peptides in B. subtilis.

Keywords

Antimicrobial peptide Plectasin Bacillus subtilis Maltose-inducible promoter SUMO 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We gratefully acknowledge financial support from the National Natural Science Foundation of China (31472104), the National Basic Research Program (2012CB124703), the National Science and Technology Support Program (2013BAD10B03), the China Agriculture Research System (CARS-36), and the Program for Universities in Heilongjiang Province (1254CGZH22).

References

  1. 1.
    Cao Y, Ma Q, Shan A, Dong N (2012) Expression in Pichia pastoris and biological activity of avian β-defensin 6 and its mutant peptide without cysteines. Protein Peptide Lett 19:1064–1070. doi: 10.2174/092986612802762660 CrossRefGoogle Scholar
  2. 2.
    Chen X, Shi J, Chen R, Wen Y, Shi Y, Zhu Z, Guo S, Li L (2014) Molecular chaperones (TrxA, SUMO, Intein, and GST) mediating expression, purification, and antimicrobial activity assays of plectasin in Escherichia coli. Biotechnol Appl Bioc. doi: 10.1002/bab.1303 Google Scholar
  3. 3.
    Chen X, Zhu F, Cao Y, Qiao S (2009) Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. Antimicrob Agents Chemother 53:3683–3689. doi: 10.1128/AAC.00251-09 PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    da Costa JP, Cova M, Ferreira R, Vitorino R (2015) Antimicrobial peptides: an alternative for innovative medicines? Appl Microbiol Biotechnol 99:2023–2040. doi: 10.1007/s00253-015-6375-x CrossRefPubMedGoogle Scholar
  5. 5.
    Dong N, Zhu X, Chou S, Shan A, Li W, Jiang J (2014) Antimicrobial potency and selectivity of simplified symmetric-end peptides. Biomaterials 35:8028–8039. doi: 10.1016/j.biomaterials.2014.06.005 CrossRefPubMedGoogle Scholar
  6. 6.
    Hancock REW, Sahl H-G (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24:1551–1557. doi: 10.1038/nbt1267 CrossRefPubMedGoogle Scholar
  7. 7.
    Hara S, Mukae H, Sakamoto N et al (2008) Plectasin has antibacterial activity and no affect on cell viability or IL-8 production. Biochem Biophys Res Commun 374:709–713. doi: 10.1016/j.bbrc.2008.07.093 CrossRefPubMedGoogle Scholar
  8. 8.
    He Q, Fu AY, Li TJ (2015) Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus subtilis. J Ind Microbiol Biotechnol 42:647–653. doi: 10.1007/s10295-014-1582-5 CrossRefPubMedGoogle Scholar
  9. 9.
    Ilk N, Schumi CT, Bohle B, Egelseer EM, Sleytr UB (2011) Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy. Microb Cell Fact. doi: 10.1186/1475-2859-10-6 PubMedCentralPubMedGoogle Scholar
  10. 10.
    Jing X, Luo X, Tian W, Lv L, Jiang Y, Wang N, Zhang T (2010) High-level expression of the antimicrobial peptide plectasin in Escherichia coli. Curr Microbiol 61(3):197–202. doi: 10.1007/s00284-010-9596-3 CrossRefPubMedGoogle Scholar
  11. 11.
    Li Y (2011) Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif 80(2):260–267. doi: 10.1016/j.pep.2011.08.001 CrossRefPubMedGoogle Scholar
  12. 12.
    Li Y (2013) Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin–SUMO dual fusion system. Protein Expr Purif 87:72–78. doi: 10.1016/j.pep.2012.10.008 CrossRefPubMedGoogle Scholar
  13. 13.
    Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ (2014) Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol 98(8):3651–3658. doi: 10.1007/s00253-013-5246-6 CrossRefPubMedGoogle Scholar
  14. 14.
    Malakhov M, Mattern M, Malakhova O, Drinker M, Weeks S, Butt T (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genom 5:75–86. doi: 10.1023/B:JSFG.0000029237.70316.52 CrossRefGoogle Scholar
  15. 15.
    Mygind PH, Fischer RL, Schnorr KM et al (2005) Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437:975–980. doi: 10.1038/nature04051 CrossRefPubMedGoogle Scholar
  16. 16.
    Parachin NdS, Mulder KC, AnArB Viana, Dias SC, OvL Franco (2012) Expression systems for heterologous production of antimicrobial peptides. Peptides 38:446–456. doi: 10.1016/j.peptides.2012.09.020 CrossRefPubMedGoogle Scholar
  17. 17.
    Reverter D, Lima Cd (2009) Preparation of SUMO proteases and kinetic analysis using endogenous substrates. SUMO Protocols, LondonCrossRefGoogle Scholar
  18. 18.
    Vavrova´ Ludmila, Muchova´ Katarı´na, Bara´k Imrich (2010) Comparison of different Bacillus subtilis expression systems. Res Microbiol 161:791–797. doi: 10.1016/j.resmic.2010.09.004 CrossRefPubMedGoogle Scholar
  19. 19.
    Wang Y, Liu Y, Wang Z, Lu F (2014) Influence of promoter and signal peptide on the expression of pullulanase in Bacillus subtilis. Biotechnol Lett 36:1783–1789. doi: 10.1007/s10529-014-1538-x CrossRefPubMedGoogle Scholar
  20. 20.
    Wu S, Zhang F, Huang Z et al (2012) Effects of the antimicrobial peptide cecropin AD on performance and intestinal health in weaned piglets challenged with Escherichia coli. Peptides 35:225–230. doi: 10.1016/j.peptides.2012.03.030 CrossRefPubMedGoogle Scholar
  21. 21.
    Yang M, Zhang W, Chen Y, Gong Y (2010) Development of a Bacillus subtilis expression system using the improved Pglv promoter. Microb Cell Fact 9:55. doi: 10.1186/1475-2859-9-55 CrossRefGoogle Scholar
  22. 22.
    Yang M, Zhang W, Ji S, Cao P, Chen Y, Zhao X (2013) Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system. PLoS One 8(2):e56321. doi: 10.1371/journal.pone.0056321 PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Yang M, Zhang W, Zhang X, Cen P (2006) Construction and characterization of a novel maltose inducible expression vector in Bacillus subtilis. Biotechnol Lett 28:1713–1718. doi: 10.1007/s10529-006-9146-z CrossRefGoogle Scholar
  24. 24.
    Yang Y, Teng D, Zhang J, Tian Z, Wang S, Wang J (2011) Characterization of recombinant plectasin: solubility, antimicrobial activity and factors that affect its activity. Process Biochem 46(5):1050–1055. doi: 10.1016/j.procbio.2011.01.018 CrossRefGoogle Scholar
  25. 25.
    Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7:620–634. doi: 10.1002/biot.201100155 CrossRefPubMedGoogle Scholar
  26. 26.
    Zhang J, Yang Y, Teng D, Tian Z, Wang S, Wang J (2011) Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphylococcus and Streptococcus. Protein Expr Purif 78:189–196. doi: 10.1016/j.pep.2011.04.014 CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2015

Authors and Affiliations

  • Licong Zhang
    • 1
  • Xiaodan Li
    • 1
  • Dandan Wei
    • 1
  • Jue Wang
    • 1
  • Anshan Shan
    • 1
    Email author
  • Zhongyu Li
    • 1
  1. 1.Institute of Animal NutritionNortheast Agricultural UniversityHarbinChina

Personalised recommendations

Cite article

Buy options