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Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3)

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Abstract

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni2+-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC50 of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.

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References

  1. Arechaga I, Miroux B, Karrasch S, Huijbregts R, de Kruijff B, Runswick MJ, Walker JE (2000) Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. FEBS Lett 482:215–219

  2. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

  3. Bradford MM (1978) A rapid and sensitive method for quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–258

  4. Cao W, Zhou Y, Ma Y, Luo Q, Wei D (2005) Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli. Protein Expr Purif 40:404–410

  5. Dathe M, Wieprecht T (1999) Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta 1462:71–87

  6. de las Rivas B, Curiel JA, Mancheño JM, Muñoz R (2007) Expression vectors for enzyme restriction- and ligation-independent cloning for producing recombinant His-fusion proteins. Biotechnol Prog 23:680–686

  7. Dumon-Seignovert L, Cariot G, Vuillard L (2004) The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expr Purif 37:203–206

  8. Feng XJ, Wang JH, Shan AS, Teng D, Yang YL, Yao Y, Yang GP, Shao YC, Liu S, Zhang F (2006) Fusion expression of bovine lactoferricin in Escherichia coli. Protein Expr Purif 47:110–117

  9. Finlay BB, Hancock RE (2007) Can innate immunity be enhanced to treat microbial infections? Nat Rev Microbiol 2:497–504

  10. Hancock RE (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1:156–164

  11. Hockney RC (1994) Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol 12:456–463

  12. 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 19:1–9

  13. Kraeva RI, Krastev DB, Roguev A, Ivanova A, Nedelcheva-Veleva MN, Stoynov SS (2007) Stability of mRNA/DNA and DNA/DNA duplexes affects mRNA transcription. PLoS ONE 2:e290

  14. Kurland CG, Dong H (1996) Bacterial growth inhibition by overproduction of protein. Mol Microbiol 21:1–4

  15. Li Y, Li X, Wang G (2006) Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. Protein Expr Purif 47:498–505

  16. Li Y, Li X, Li H, Lockridge O, Wang G (2007) A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation. Protein Expr Purif 54:157–165

  17. Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S (2005) Comparison of affinity tags for protein purification. Protein Expr Purif 41:98–105

  18. 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 171:2166–2172

  19. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298

  20. Mitsuda Y, Takimoto A, Kamitani S, Kitamura K, Sakata T, Mitsushima K (2002) Large-scale production of functional human adrenomedullin: expression, cleavage, amidation, and purification. Protein Expr Purif 25:448–455

  21. Pütsep K, Brändén CI, Boman HG, Normark S (1999) Antibacterial peptide from H. pylori. Nature 398:671–672

  22. Rabhi-Essafi I, Sadok A, Khalaf N, Fathallah DM (2007) A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli. Protein Eng Des Sel 20:201–209

  23. Rao XC, Li S, Hu JC, Jin XL, Hu XM, Huang JJ, Chen ZJ, 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

  24. Sørensen HP, Sperling-Petersen HU, Mortensen KK (2003) Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck. J Chromatogr B Analyt Technol Biomed Life Sci 786:207–214

  25. Strøm MB, Haug BE, Rekdal O, Skar ML, Stensen W, Svendsen JS (2002) Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity. Biochem Cell Biol 80:65–74

  26. Thai K, Choi J, Franzin CM, Marassi FM (2005) Bcl-XL as a fusion protein for the high-level expression of membrane-associated proteins. Protein Sci 14:948–955

  27. Tian ZG, Teng D, Yang YL, Luo J, Feng XJ, Fan Y, Zhang F, Wang JH (2007) Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli. Appl Microbiol Biotechnol 75:117–124

  28. Tian ZG, Dong TT, Teng D, Yang YL, Wang JH (2009) Design and characterization of novel antimicrobial peptides based on structure parameters by computer-aided method. Appl Microbiol Biotechnol. doi:https://doi.org/10.1007/s00253-008-1839-x

  29. Wang Z, Wang G (2004) APD: the Antimicrobial Peptide Database. Nucleic Acids Res 32:D590–D592

  30. Wang L, Wu H, Dou F, Xie W, Xu X (1997) High-level expression of cecropin CMIV in E. coli from a fusion construct containing the human tumor necrosis factor. Biochem Mol Biol Int 41:1051–1056

  31. Xu X, Jin F, Yu X, Ren S, Hu J, Zhang W (2007) High-level expression of the recombinant hybrid peptide cecropinA(1–8)–magainin2(1–12) with an ubiquitin fusion partner in Escherichia coli. Protein Expr Purif 55:175–182

  32. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–56

  33. Zhou QF, Luo XG, Ye L, Xi T (2007) High-level production of a novel antimicrobial peptide perinerin in Escherichia coli by fusion expression. Curr Microbiol 54:366–370

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Acknowledgments

This study is supported by National Natural Science Foundation of China (no. 30771574; no. 30810303084), Beijing Natural Science Foundation (no. 5062031; no. 5093030) and Chinese 863 Program (no. 2004AA246040).

Author information

Correspondence to Jian-hua Wang.

Additional information

Zi-gang Tian and Tian-tang Dong contributed equally to this paper.

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Tian, Z., Dong, T., Yang, Y. et al. Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3). Appl Microbiol Biotechnol 83, 143–149 (2009). https://doi.org/10.1007/s00253-009-1893-z

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Keywords

  • Antimicrobial peptide
  • Expression
  • Multimerization
  • Escherichia coli
  • Formic acid