Skip to main content

Advertisement

SpringerLink
Log in

High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage

  • Applied Genetics and Molecular Biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Cipakova I, Hostinova E, Gasperik J, Velebny V (2004) High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli. Protein Expr Purif 37:207–212

    Article  CAS  Google Scholar 

  • Creemers JW, Usac EF, Bright NA, Van de Loo JW, Jansen E, Van de Ven WJ, Hutton JC (1996) Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. J Biol Chem 271:25284–25291

    Article  CAS  Google Scholar 

  • Dhople V, Krukemeyer A, Ramamoorthy A (2006) The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochim Biophys Acta 1758:1499–1512

    Article  CAS  Google Scholar 

  • Dürr UH, Sudheendra US, Ramamoorthy A (2006) LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim Biophys Acta 1758:1408–1425

    Article  Google Scholar 

  • Hancock RE, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24:1551–1557

    Article  CAS  Google Scholar 

  • Ingham AB, Moore RJ (2007) Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol Appl Biochem 47:1–9

    Article  CAS  Google Scholar 

  • Iost I, Dreyfus M (1995) The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. EMBO J 14:3252–3261

    Article  CAS  Google Scholar 

  • Jenssen H, Hamill P, Hancock RE (2006) Peptide antimicrobial agents. Clin Microbiol Rev 19:491–511

    Article  CAS  Google Scholar 

  • Kim HK, Chun DS, Kim JS, Yun CH, Lee JH, Hong SK, Kang DK (2006) Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli. Appl Microbiol Biotechnol 72:330–338

    Article  CAS  Google Scholar 

  • Lee JH, Minn I, Park CB, Kim SC (1998) Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli. Protein Expr Purif 12:53–60

    Article  CAS  Google Scholar 

  • Lee JH, Kim JH, Hwang SW, Lee WJ, Yoon HK, Lee HS, Hong SS (2000) High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem Biophys Res Commun 277:575–580

    Article  CAS  Google Scholar 

  • Lee JH, Kim MS, Cho JH, Kim SC (2002) Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant. Appl Microbiol Biotechnol 58:790–796

    Article  CAS  Google Scholar 

  • Li L, Wang JX, Zhao XF, Kang CJ, Liu N, Xiang JH, Li FH, Sueda S, Kondo H (2005) High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expr Purif 39:144–151

    Article  CAS  Google Scholar 

  • Makovitzki A, Avrahami D, Shai Y (2006) Ultrashort antibacterial and antifungal lipopeptides. Proc Natl Acad Sci U S A 103:15997–16002

    Article  CAS  Google Scholar 

  • Mecke A, Lee DK, Ramamoorthy A, Orr BG, Banaszak Holl MM (2005) Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers. Biophys J 89:4043–4050

    Article  CAS  Google Scholar 

  • Merrifield RB (1963) Solid phase peptide synthesis, I, the synthesis of a tetrapeptide. J Am Chem Soc 85:2149–2154

    Article  CAS  Google Scholar 

  • Molloy SS, Bresnahan PA, Leppla SH, Klimpel KR, Thomas G (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402

    CAS  PubMed  Google Scholar 

  • Mookherjee N, Hancock RE (2007) Cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections. Cell Mol Life Sci 64:922–933

    Article  CAS  Google Scholar 

  • Moon JY, Henzler-Wildman KA, Ramamoorthy A (2006) Expression and purification of a recombinant LL-37 from Escherichia coli. Biochim Biophys Acta 1758:1351–1358

    Article  CAS  Google Scholar 

  • Morassutti C, Amicis FD, Bandiera A, Marchetti S (2005) Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system. Protein Expr Purif 39:160–168

    Article  CAS  Google Scholar 

  • Morin KM, Arcidiacono S, Beckwitt R, Mello CM (2006) Recombinant expression of indolicidin concatamers in Escherichia coli. Appl Microbiol Biotechnol 70:698–704

    Article  CAS  Google Scholar 

  • Park CB, Yi KS, Matsuzaki K, Kim MS, Kim SC (2000) Structure–activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II. Proc Natl Acad Sci U S A 97:8245–8250

    Article  CAS  Google Scholar 

  • Park IY, Cho JH, Kim KS, Kim YB, Kim MS, Kim SC (2004) Helix stability confers salt resistance upon helical antimicrobial peptides. J Biol Chem 279:13896–13901

    Article  CAS  Google Scholar 

  • Porcelli F, Buck-Koehntop BA, Thennarasu S, Ramamoorthy A, Veglia G (2006) Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy. Biochemistry 45:5793–5799

    Article  CAS  Google Scholar 

  • Ramamoorthy A, Thennarasu S, Lee DK, Tan A, Maloy L (2006) Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin. Biophys J 91:206–216

    Article  CAS  Google Scholar 

  • Rao XC, Li S, Hu JC, Jin XL, Hu XM, Huang JJ, Chen JG, Zhu JM, Hu FQ (2004) A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli. Protein Expr Purif 36:11–18

    Article  CAS  Google Scholar 

  • Reddy KV, Yedery RD, Aranda C (2004) Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 24:536–547

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Szynol A, de Haard JJ, Veerman EC, de Soet JJ, van Nieuw Amerongen AV (2006) Design of a peptibody consisting of the antimicrobial peptide dhvar5 and a llama variable heavy-chain antibody fragment. Chem Biol Drug Des 67:425–431

    Article  CAS  Google Scholar 

  • Tanaka T, Yada RY (1996) Expression of soluble cloned porcine pepsinogen A in Escherichia coli. Biochem J 315:443–446

    Article  CAS  Google Scholar 

  • Wei Q, Kim YS, Seo JH, Jang WS, Lee IH, Cha HJ (2005) Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli. Appl Environ Microbiol 71:5038–5043

    Article  CAS  Google Scholar 

  • Xu X, Jin F, Yu X, Ji S, Wang J, Cheng H, Wang C, Zhang W (2007) Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr Purif 53:293–301

    Article  CAS  Google Scholar 

  • Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported, in part, by grants from 21C Frontier Program of Microbial Genomics and Applications (MG05-0204-1-0), the Molecular and Cellular BioDiscovery Research Program (M1-0106-00-0200) from the Ministry of Science and Technology of Korea, the Korea Science and Engineering Foundation Grant (R01-2005-000-11010-0), and the Basic Research Program of the Korea Research Foundation Grant (KRF-2004-042-D00072).

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, South Korea

    Jung Min Kim, Su A Jang, Byung Jo Yu, Bong Hyun Sung & Sun Chang Kim

  2. Department of Biology, Gyeongsang National University, Jinju, 660-701, South Korea

    Ju Hyun Cho

  3. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon, 305-701, South Korea

    Sun Chang Kim

Authors
  1. Jung Min Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Su A Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Byung Jo Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bong Hyun Sung
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ju Hyun Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sun Chang Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sun Chang Kim.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, J.M., Jang, S.A., Yu, B.J. et al. High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl Microbiol Biotechnol 78, 123–130 (2008). https://doi.org/10.1007/s00253-007-1273-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-007-1273-5

Keywords

Search

Navigation