Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Expression of Recombinant Hybrid Peptide Gaegurin4 and LL37 using Fusion Protein in E. coli

Glutathione S-Transferase에 융합한 재조합 Hybrid Peptide Gaegurin-LL37의 대장균에서의 발현

  • Bayarbat, Ishvaanjil (Department of Chemistry, School of Natural Science, Mongolian State University of Agriculture) ;
  • Lee, Jae-Hag (Department of Food and Nutrition, Seoil College) ;
  • Lee, Soon-Youl (Department of Biotechnology, Genetic Engineering Research Institute, Hankyong National University)
  • Received : 2012.03.13
  • Accepted : 2012.04.03
  • Published : 2012.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.

항균 펩타이드(Antimicrobial peptides(AMPs)는 그람 양성, 그람 음성 세균과 진균병원체에 대항하는 생명체에서 중요한 역할을 하는 물질이다. 인간의 Cathelicidin 항균 펩타이드는 임상학적으로 사용할 수 있는 여러 가지의 생물학적 활성을 가진다. 항균 펩타이드의 생산 비용은 재조합 방법으로 낮출 수가 있다. 대장균은 저렴하며 손쉬운 조작이 가능하기 때문에 다른 재조합 단백질처럼 항균 펩타이드의 발현에 훌륭한 숙주가 될 수 있다. 그러나 대장균에서의 항균 펩타이드의 과발현은 항균 펩타이드가 과발현 되었을 때 대장균에 독성을 보일 수 있으므로 어려움이 보고가 되어있다. 본 연구에서는 이러한 문제점을 극복하고자 항균 펩타이드를 Glutathione S-transferase(GST) 결합 단백질에 융합하여 항균펩타이드의 독성을 감소시키도록 설계하여 발현을 시도하였다. 이 때 발현한 항균 펩티드는 LL37과, gaegurin4과 LL37의 잡종 펩타이드 GGN4-LL37(GL32로 명명)를 GST에 융합되도록 벡터를 구축하고 설계하여 대장균에서 GST 융합단백질로 발현시켰다. 융합 단백질은 친화력 컬럼을 사용하여 분리하고 GST를 절단하여 항균펩타이드 만을 분리하였고 분리한 펩타이드는 웨스턴 블롯팅으로 확인하였고 그람 양성, 그람 음성 세균에 대하여 항균 활성을 나타내는 것을 확인하였다.

Keywords

References

  1. Andersson, E., O. E. Sorensen, B. Frohm, N. Borregaard, A. Egesten, and J. Malm. 2002. Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes. Hum. Reprod. 10: 2529-2534.
  2. Bals, R. 2000. Epithelial antimicrobial peptides in host defense against infection. Respir. Res. 1: 141-150.
  3. Boman, H. G., I. Faye, G. H. Gudmundsson, J. Y. Lee, and D. A. Lid-holm. 1991. Cell free immunity in Cecropia. A model system for antibacterial proteins. Eur. J. Biochem. 210: 23-31.
  4. Boman, H. G. 2003. Antibacterial peptides: basic facts and emerging concepts. J. Intern. Med. 254:197-215. https://doi.org/10.1046/j.1365-2796.2003.01228.x
  5. Frasca, L. and R. Lande. 2012. Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases. Curr. Pharm. Biotechnol. [Epub ahead of print].
  6. Frohm, M., B. Agerberth, G. Ahangari, M. Stahle-Backdahl, S. Liden, H. Wigzell, and G. H. Gudmundsson. 1997. The expression of the gene coding for the antibacterial peptide LL37 is induced in human keratinocytes during inflammatory disorders. J. Biol. Chem. 272: 15258-15263. https://doi.org/10.1074/jbc.272.24.15258
  7. Harder, J. J., Bartels, E., Christophers, and J. M. Schroder. 1997. A peptide antibiotic from human skin. Nature 387: 861-862. https://doi.org/10.1038/43088
  8. Ingham, A. B. and R. J. Moore. 2007. Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol. Appl. Biochem. 47: 1-9. https://doi.org/10.1042/BA20060207
  9. Jing, X. L., X. G. Luo, W. J. Tian, L. H. Lv, Y. Jiang, N. Wang, and T. C. Zhang. 2010. High-level expression of the antimicrobial peptide plectasin in Escherichia coli. Curr. Microbiol. 61: 197-202. https://doi.org/10.1007/s00284-010-9596-3
  10. Krahulec, J, M. Hyrsova, S. Pepeliaev, J. Jílkova, Z. Cerny, and J. Machalkova. 2010. High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli. Appl. Microbiol. Biotechnol. 88: 167-175. https://doi.org/10.1007/s00253-010-2736-7
  11. Kwon, S. Y., B. A. Carlson, J. M. Park, and B. J. Lee. 2000. Structural organization and expression of the gaegurin 4 gene of Rana rugosa. Biochim. Biophys. Acta. 1492: 185-190. https://doi.org/10.1016/S0167-4781(00)00082-8
  12. Lai, Y. and R. L. Gallo. 2009. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends. Immunol. 30: 131-141. https://doi.org/10.1016/j.it.2008.12.003
  13. Miyasaki, K. T. and R. I. Lehrer. 1998. $\beta$-sheet antibiotic peptides as potential dental therapeutics. Int. J. Antimicrob. Agents. 9: 269-280. https://doi.org/10.1016/S0924-8579(98)00006-5
  14. Nijnik, A. and R. E. Hancock. 2009. The roles of cathelicidin LL-37 in immune defences and novel clinical applications. Curr. Opin. Hematol. 16:41-47. https://doi.org/10.1097/MOH.0b013e32831ac517
  15. Park, J. M., J. E. Jung, and B. J. Lee. 1994. Antimicrobial peptides from the skin of a Korean frog, Rana Rugosa. Biochem. Biophy. Res. Commun. 205: 948-954. https://doi.org/10.1006/bbrc.1994.2757
  16. Ramos, R., L. Domingues, and M. Gama. 2010. Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein Expr. Purif. 71: 1-7. https://doi.org/10.1016/j.pep.2009.10.016
  17. Smith, R. D. and J. Coast. 2002. Antimicrobial resistance: a global response. Bull. World Health Organ. 80: 126-133.
  18. Tsai, H. and L. A. Bobek. 1998. Human salivary histatins: promising anti-fungal therapeutic agents. Crit. Rev. Oral Biol. Med. 9: 480-497. https://doi.org/10.1177/10454411980090040601
  19. Turner, J., Y. Cho, N. N. Dinh, A. J. Waring, and R. I. Lehrer. 1998. Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob. Agents. Chemother. 42: 2206-2214.
  20. Wiesner, J. and A. Vilcinskas. 2010. Antimicrobial peptides, The ancient arm of the human immune system. Virulence 1: 440-464. https://doi.org/10.4161/viru.1.5.12983
  21. Wilmes, M., B. P. Cammue, H. G. Sahl, and K. Thevissen. 2011. Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation. Nat. Prod. Rep. 28: 1350-1358. https://doi.org/10.1039/c1np00022e
  22. Yang, Y. H., G. G. Zheng, G. Li, X. J. Zhang, Z. Y. Cao, R. Qing, and K. F. Wu. 2004. Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL37, in E.coli. Protein Expr. Purif. 37: 229-235. https://doi.org/10.1016/j.pep.2004.06.007
  23. Zasloff, M. 2002. Antimicrobial peptides of muticellular organisms. Nature 415: 389-395. https://doi.org/10.1038/415389a

Cited by

  1. Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity vol.26, pp.23, 2012, https://doi.org/10.3390/molecules26237142