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Applied Microbiology and Biotechnology

, Volume 98, Issue 8, pp 3651–3658 | Cite as

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology

  • Chao Luan
  • Hai Wen Zhang
  • De Guang Song
  • Yong Gang Xie
  • Jie Feng
  • Yi Zhen WangEmail author
Biotechnologically relevant enzymes and proteins

Abstract

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.

Keywords

Small ubiquitin-related modifier SUMO protease 1 Bacillus subtilis Cathelicidin-BF Antimicrobial peptide 

Notes

Acknowledgments

This work was financially supported by the Modern Agro-Industry Technology Research System (no. CARS-36) and 12th Five-year Plan-National Science and Technology Programs in Agricultural Areas (2011BAD26B002-5).

References

  1. Andersson DI, Hughes D (2010) Antibiotic resistance and its cost: is it possible to reverse resistance? Nat Rev Microbiol 8(4):260–271PubMedGoogle Scholar
  2. 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 10:6PubMedCentralPubMedCrossRefGoogle Scholar
  3. Kakeshita H, Kageyama Y, Endo K, Tohata M, Ara K, Ozaki K, Nakamura K (2011) Secretion of biologically-active human interferon-β by Bacillus subtilis. Biotechnol Lett 33(9):1847–1852PubMedCrossRefGoogle Scholar
  4. Leach CA, Tian X, Mattern MR, Nicholson B (2009) Detection and characterization of SUMO protease activity using a sensitive enzyme-based reporter assay. Methods Mol Biol 497:269–281PubMedCrossRefGoogle Scholar
  5. Li W, Zhou X, Lu P (2004) Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol 155(8):605–610PubMedCrossRefGoogle Scholar
  6. Li Y (2011) Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif 80(2):260–267PubMedCrossRefGoogle Scholar
  7. Lowe AJ, Bardliving CL, Batt CA (2012) Methods for chromatographic removal of endotoxin. Methods Mol Biol 899:265–275PubMedCrossRefGoogle Scholar
  8. Magalhaes PO, Lopes AM, Mazzola PG, Rangel-Yagui C, Penna TC, Pessoa A Jr (2007) Methods of endotoxin removal from biological preparations: a review. J Pharm Pharm Sci 10(3):388–404PubMedGoogle Scholar
  9. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics 5(1–2):75–86PubMedCrossRefGoogle Scholar
  10. Reverter D, Lima CD (2009) Preparation of SUMO proteases and kinetic analysis using endogenous substrates. Methods Mol Biol 497:225–239PubMedCentralPubMedCrossRefGoogle Scholar
  11. Satakarni M, Curtis R (2011) Production of recombinant peptides as fusions with SUMO. Protein Expr Purif 78(2):113–119PubMedCrossRefGoogle Scholar
  12. Sun Z, Xia Z, Bi F, Liu JN (2008) Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli. Appl Microbiol Biotechnol 78(3):495–502PubMedCrossRefGoogle Scholar
  13. Takesue N, Sone T, Tanaka M, Tomita F, Asano K (2009) Effect of an additionally introduced degQ gene on di-D-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system. J Biosci Bioeng 107(6):623–629PubMedCrossRefGoogle Scholar
  14. Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U (2011) A flow cytometry-based screening system for directed evolution of proteases. J Biomol Screen 16(3):285–294PubMedCrossRefGoogle Scholar
  15. Wang Y, Hong J, Liu X, Yang H, Liu R, Wu J, Wang A, Lin D, Lai R (2008) Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics. PLoS One 3(9):e3217PubMedCentralPubMedCrossRefGoogle Scholar
  16. Wang Y, Zhang Z, Chen L, Guang H, Li Z, Yang H, Li J, You D, Yu H, Lai R (2011) Cathelicidin-BF, a snake cathelicidin-derived antimicrobial peptide, could be an excellent therapeutic agent for acne vulgaris. PLoS One 6(7):e22120PubMedCentralPubMedCrossRefGoogle Scholar
  17. Wenzel M, Muller A, Siemann-Herzberg M, Altenbuchner J (2011) Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation. Appl Environ Microbiol 77(18):6419–6425PubMedCentralPubMedCrossRefGoogle Scholar
  18. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694(1–3):299–310PubMedCrossRefGoogle Scholar
  19. Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3(2):163–175PubMedCrossRefGoogle Scholar
  20. Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173(16):4952–4958PubMedCentralPubMedGoogle Scholar
  21. Yeh CM, Yeh CK, Hsu XY, Luo QM, Lin MY (2008) Extracellular expression of a functional recombinant Ganoderma lucidium immunomodulatory protein by Bacillus subtilis and Lactococcus lactis. Appl Environ Microbiol 74(4):1039–1049PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Chao Luan
    • 1
  • Hai Wen Zhang
    • 1
  • De Guang Song
    • 1
  • Yong Gang Xie
    • 1
  • Jie Feng
    • 1
  • Yi Zhen Wang
    • 1
    Email author
  1. 1.Key Laboratory of Animal Nutrition and Feed Science, Ministry of Agriculture (East China), Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Institute of Feed ScienceZhejiang UniversityHangzhouPeople’s Republic of China

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