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Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Antimicrobial peptides (AMPs) have been paid considerable attention owing to their broad-spectrum antimicrobial activity and have great potential as novel antimicrobials. In this study, a novel hybrid peptide LF15-CA8 was designed on the basis of bovine lactoferricin (LfcinB) and cecropin A. The gene segment encoding LF15-CA8 was synthesized and cloned into pGEX-4T-BH to form pGEX-4T-LC1 containing one copy of the LF15-CA8 coding region. A series of recombinant vectors containing up to six multiple-copy LF15-CA8 coding regions, i.e., pGEX-4T-LCn (n = 1–6), were subsequently constructed, and used for transformation in Escherichia coli BL21(DE3). After induction with IPTG, pGEX-4T-LC1 and pGEX-4T-LC2 transformants successfully expressed fusion proteins GST-LF15-CA8 and GST-(LF15-CA8)2 in the form of inclusion bodies, respectively. The inclusion bodies were dissolved and the peptide was successfully released in 70 % formic acid in a single step. After purification, about 10.0 mg of the recombinant peptide LF15-CA8 with purity more than 97 % was obtained from 1 l of bacteria culture of pGEX-4T-LC2 transformants. LF15-CA8 caused an increase in antibacterial activity against Gram-positive bacterium (Staphylococcus aureus ATCC 25923) compared with the parent peptides and did not show obvious hemolytic activity against human erythrocytes in the range of effective antibacterial concentration. These results suggest that the peptide LF15-CA8 could be a promising candidate for therapeutic applications, and may lead to a cost-effective solution for the large-scale production of AMPs.

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References

  1. Andreu D, Ubach J, Boman A, Wåhlin B, Wade D, Merrifield RB, Boman HG (1992) Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity. FEBS Lett 296(2):190–194

    Article  CAS  PubMed  Google Scholar 

  2. Bang SK, Kang CS, Han MD, Bang IS (2010) Expression of recombinant hybrid peptide Hinnavin II/aIpha-melanocyte-stimulating hormone in Escherichia coli: purification and characterization. J Microbiol 48(1):24–29

    Article  CAS  PubMed  Google Scholar 

  3. Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M (1992) Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol 73(6):472–479

    Article  CAS  PubMed  Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  5. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3(3):238–250

    Article  CAS  PubMed  Google Scholar 

  6. Cao Y, Yu RQ, Liu Y, Zhou HX, Song LL, Cao Y, Qiao DR (2010) Design, recombinant expression, and antibacterial activity of the cecropins-melittin hybrid antimicrobial peptides. Curr Microbiol 61(3):169–175

    Article  CAS  PubMed  Google Scholar 

  7. Chen ZJ, Wang DM, Cong YG, Wang J, Zhu JM, Yang J, Hu Z, Hu XM, Tan YL, Hu FQ, Rao XC (2011) Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol 89(2):281–291

    Article  CAS  PubMed  Google Scholar 

  8. Ferre R, Melo MN, Correia AD, Feliu L, Bardají E, Planas M, Castanho M (2009) Synergistic effects of the membrane actions of cecropin-melittin antimicrobial hybrid peptide BP100. Biophys J 96(5):1815–1827

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Gifford JL, Hunter HN, Vogel HJ (2005) Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci 62(22):2588–2598

    Article  CAS  PubMed  Google Scholar 

  10. Guani-Guerra E, Santos-Mendoza T, Lugo-Reyes SO, Teran LM (2010) Antimicrobial peptides: general overview and clinical implications in human health and disease. Clin Immunol 135(1):1–11

    Article  CAS  PubMed  Google Scholar 

  11. Hultmark D, Steiner H, Rasmuson T, Boman HG (1980) Insect immunity, purification and properties of three inducible bactericidal proteins from emolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem 106(1):7–16

    Article  CAS  PubMed  Google Scholar 

  12. Iost I, Dreyfus M (1995) The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. EMBO J 14(13):3252–3261

    CAS  PubMed  Google Scholar 

  13. Jang SA, Kim H, Lee JY, Shin JR, da Kim J, Cho JH, Kim SC (2012) Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb. Peptides 34(2):283–289

    Article  CAS  PubMed  Google Scholar 

  14. Jonasson P, Nygren PA, Jornvall H, Johansson BL, Wahren J, Uhlen M, Stahl S (2000) Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein. J Biotechnol 76(2–3):215–226

    Article  CAS  PubMed  Google Scholar 

  15. Khan J, Gupta S, Chattopadhyay K, Mukhopadhaya A (2012) Refolding and functional assembly of the Vibrio cholerae porin OmpU recombinantly expressed in the cytoplasm of Escherichia coli. Protein Expr Purif 85(2):204–210

    Article  CAS  PubMed  Google Scholar 

  16. Kim JM, Jang SA, Yu BJ, Sung BH, Cho JH, Kim SC (2008) High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl Microbiol Biotechnol 78(1):123–130

    Article  CAS  PubMed  Google Scholar 

  17. Kovaleva V, Krynytskyy H, Gout I, Gout R (2011) Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1. Appl Microbiol Biotechnol 89(4):1093–1101

    Article  CAS  PubMed  Google Scholar 

  18. Lee DG, Park Y, Jin I, Hahm KS, Lee HH, Moon YH, Woo ER (2004) Structure-antiviral activity relationships of cecropin A-magainin 2 hybrid peptide and its analogues. J Pept Sci 10(5):298–303

    Article  CAS  PubMed  Google Scholar 

  19. 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(6):790–796

    Article  CAS  PubMed  Google Scholar 

  20. Lee M, Bang K, Kwon H, Cho S (2013) Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker. Mol Biol Rep 40(6):3953–3960

    Article  CAS  PubMed  Google Scholar 

  21. Mader JS, Salsman J, Conrad DM, Hoskin DW (2005) Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines. Mol Cancer Ther 4(4):612–624

    Article  CAS  PubMed  Google Scholar 

  22. Maher S, McClean S (2006) Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochem Pharmacol 71(9):1289–1298

    Article  CAS  PubMed  Google Scholar 

  23. Miller KW, Evans RJ, Eisenberg SP, Thompson RC (1989) Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli. J Bacteriol 171(4):2166–2172

    CAS  PubMed Central  PubMed  Google Scholar 

  24. Moore AJ, Beazley WD, Bibby MC, Devine DA (1996) Antimicrobial activity of cecropins. J Antimicrob Chemother 37(6):1077–1089

    Article  CAS  PubMed  Google Scholar 

  25. Park TJ, Kim JS, Choi SS, Kim Y (2009) Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli. Protein Expr Purif 65(1):23–29

    Article  CAS  PubMed  Google Scholar 

  26. Paulsen VS, Blencke HM, Benincasa M, Haug T, Eksteen JJ, Styrvold OB, Scocchi M, Stensvåg K (2013) Structure-activity relationships of the antimicrobial peptide arasin 1 and mode of action studies of the N-terminal, proline-rich region. PLoS ONE 8(1):e53326

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Sambrook J, Fristsch EF, Maniatis T (1989) In: Sambrook J (ed) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, pp 96–99

  28. Schagger H, von Jagow G (1987) Tricine–sodium dodecyl sulfate–polyacrylamide gel electroporesis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166(2):368–379

    Article  CAS  PubMed  Google Scholar 

  29. Shen Y, Ai HX, Song R, Liang ZN, Li JF, Zhang SQ (2010) Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology. Microbiol Res 165(8):713–718

    Article  CAS  PubMed  Google Scholar 

  30. Spížek J, Novotná J, Rezanka T, Demain AL (2010) Do we need new antibiotics? The search for new targets and new compounds. J Ind Microbiol Biotechnol 37(12):1241–1248

    Article  PubMed  Google Scholar 

  31. Tian ZG, Dong TT, Yang YL, Teng D, Wang JH (2009) Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3). Appl Microbiol Biotechnol 83(1):143–149

    Article  CAS  PubMed  Google Scholar 

  32. Wang LN, Yu B, Han GQ, He J, Chen DW (2010) Design, expression and characterization of recombinant hybrid peptide Attacin–Thanatin in Escherichia coli. Mol Biol Rep 37(7):3495–3501

    Article  CAS  PubMed  Google Scholar 

  33. Wiesner J, Vilcinskas A (2010) Antimicrobial peptides: the ancient arm of the human immune system. Virulence 1(5):440–464

    Article  PubMed  Google Scholar 

  34. Wu GQ, Deng XP, Li XF, Wang XY, Wang SL, Xu HM (2011) Application of immobilized thrombin for production of S-thanatin expressed in Escherichia coli. Appl Microbiol Biotechnol 92(1):85–93

    Article  CAS  PubMed  Google Scholar 

  35. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 414(6870):389–395

    Article  Google Scholar 

  36. Zhang JL, Peng S, Cheng X, Wang HY (2012) Functional analysis of hybrid peptide CAMA-Syn: expression in mammalian cells and antimicrobial potential. Protein Pept Lett 19(10):1076–1081

    Article  CAS  PubMed  Google Scholar 

  37. Zhao PW, Cao GF (2012) Production of bioactive sheep β-defensin-1 in Pichia pastoris. J Ind Microbiol Biotechnol 39(12):11–17

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (30800794), Program for Young Aged Academic Staff in the Heilongjiang Province Ordinary College (1252G010), and Research Fund for Innovation Talents of Science and Technology in Harbin City (2012RFQXN022).

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Authors and Affiliations

  1. College of Animal Science and Technology, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin, 150030, China

    Xing-Jun Feng, Li-Wei Xing, Di Liu, Xue-Ying Song, Jing Li & Wen-Shan Xu

  2. Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 138 Haping Road, Nangang District, Harbin, 150081, China

    Chun-Long Liu

  3. Animal Husbandry Research Centre of Heilongjiang Academy of Agricultural Science, Harbin, 150086, China

    Zhong-Qiu Li

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  1. Xing-Jun Feng
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  2. Li-Wei Xing
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  3. Di Liu
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Correspondence to Xing-Jun Feng or Chun-Long Liu.

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Feng, XJ., Xing, LW., Liu, D. et al. Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli . J Ind Microbiol Biotechnol 41, 527–534 (2014). https://doi.org/10.1007/s10295-013-1382-3

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  • DOI: https://doi.org/10.1007/s10295-013-1382-3

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