Applied Microbiology and Biotechnology

, Volume 103, Issue 4, pp 1765–1775 | Cite as

Design, expression, and characterization of a novel cecropin A-derived peptide with high antibacterial activity

  • Meng Wang
  • Jinglian Lin
  • Qiuli Sun
  • Kaiwen Zheng
  • Yi Ma
  • Jufang WangEmail author
Biotechnologically relevant enzymes and proteins


In recent years, antimicrobial peptides have received increased interest and are potential substitutes for antibiotics. However, natural antimicrobial peptides are always toxic to mammalian cells and usually exhibit weak antibacterial activity, which restrict their wide application. In this study, a novel antibacterial peptide named PEW300 was designed with three mutations to the parental peptide cecropin A. As predicted by bioinformatic programs, the positive charge of PEW300 increased from + 6 to + 9 compared with cecropin A, and the grand average of hydropathicity increased from − 0.084 to − 0.008. Expression of PEW300 resulted in serious inhibition of Escherichia coli BL21(DE3) cells, indicating designed PEW300 may have stronger antibacterial activity. A simple, fast, and low-cost approach without tedious protein purification steps was selected for the efficient production of PEW300 by fusion with ELK16 and about 7.38 μg/mg wet cell weight PEW300 was eventually obtained. Purified PEW300 exhibited strong antibacterial activity against various Gram-positive and Gram-negative bacteria which was enhanced four- to sevenfold compared with the parental peptide cecropin A. Besides, PEW300 had no hemolytic activity toward mammalian cells even at high concentration (224 ng/μl). PEW300 showed good stability in neutral and alkaline solutions. Moreover, PEW300 was thermally stable even at up to 100 °C and resistant to proteinase K, pepsin, snailase, and trypsin. The incubation with human serum had no effect on the antibacterial activity of PEW300. All these results demonstrated that PEW300 designed in this work may have good potential as a candidate pharmaceutical agent.


Antimicrobial peptide Escherichia coli Cecropin A PEW300 Prokaryotic expression 



This study was supported by the Innovative Program of Department of Education of Guangdong Province, China (grant number 2013KJCX0013) and Natural Science Foundation of Guangdong Province, China (grant number 2015A030310322).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

We state this is not applicable. The manuscript does not contain data from any individual person.

Supplementary material

253_2018_9592_MOESM1_ESM.pdf (314 kb)
ESM 1 (PDF 313 kb)


  1. And DA, Shai Y (2002) Conjugation of a magainin analogue with lipophilic acids controls hydrophobicity, solution assembly, and cell selectivity. Biochemistry 41(7):2254Google Scholar
  2. Arakawa Y (2015) Global spread of multidrug-resistant microbes including CRE and clinical alerts. J Chemother 63(2):187–197Google Scholar
  3. Brogden NK, Brogden KA (2011) Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? Int J Antimicrob Agents 38(3):217–225Google Scholar
  4. Cao X, Zhang Y, Mao R, Teng D, Wang X, Wang J (2015) Design and recombination expression of a novel plectasin-derived peptide MP1106 and its properties against Staphylococcus aureus. Appl Microbiol Biotechnol 99(6):2649–2662Google Scholar
  5. Chung PY, Khanum R (2017) Antimicrobial peptides as potential anti-biofilm agents against multidrug-resistant bacteria. J Microbiol Immunol Infect 50(4):405–410Google Scholar
  6. Coffelt SB, Marini FC, Watson K, Zwezdaryk KJ, Dembinski JL, Lamarca HL, Tomchuck SL, Honer zBK, Danka ES, Henkle SL (2009) The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells. Proc Natl Acad Sci U S A 106(10):3806–3811Google Scholar
  7. Da Costa JP, Cova M, Ferreira R, Vitorino R (2015) Antimicrobial peptides: an alternative for innovative medicines?. Appl Microbiol Biotechnology 99(5):2023–2040Google Scholar
  8. Datta A, Ghosh A, Airoldi C, Sperandeo P, Mroue KH, Jiménezbarbero J, Kundu P, Ramamoorthy A, Bhunia A (2015) Antimicrobial peptides: insights into membrane permeabilization, lipopolysaccharide fragmentation and application in plant disease control. Sci Rep 5:11951Google Scholar
  9. Datta A, Kundu P, Bhunia A (2016) Designing potent antimicrobial peptides by disulphide linked dimerization and N-terminal lipidation to increase antimicrobial activity and membrane perturbation: structural insights into lipopolysaccharide binding. J Colloid Interface Sci 461:335–345Google Scholar
  10. Dejaco C, Harrer M, Waldhoer T, Miehsler W, Vogelsang H, Reinisch W (2003) Antibiotics and azathioprine for the treatment of perianal fistulas in Crohn's disease. Aliment Pharmacol Ther 18(11–12):1113–1120Google Scholar
  11. Du Y, Chen B (2010) Recent advances in enantioseparation by capillary electrophoresis using antibiotics and polysaccharides as chiral selectors: a review. Chim Oggi 28(5):37–42Google Scholar
  12. Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462(1–2):11–28Google Scholar
  13. Fjell CD, Hiss JA, Hancock RE, Schneider G (2012) Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov 11(1):37–51Google Scholar
  14. Gregory SM, Cavenaugh A, Journigan V, Pokorny A, Almeida PFF (2008) A quantitative model for the all-or-none permeabilization of phospholipid vesicles by the antimicrobial peptide cecropin A. Biophys J 94(5):1667–1680Google Scholar
  15. Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16(2):82–88Google Scholar
  16. Holak TA, Engstroem A, Kraulis PJ, Lindeberg G, Bennich H, Jones TA, Gronenborn AM, Clore GM (1988) The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study. Biochemistry 27(20):7620–7629Google Scholar
  17. Hong RW, Shchepetov M, Weiser JN, Axelsen PH (2003) Transcriptional profile of the Escherichia coli response to the antimicrobial insect peptide cecropin A. Antimicrob Agents Chemother 47(1):1–6Google Scholar
  18. Huang Z, Liao F, Zheng Q, Huang Y (2001) Insect cecropin used as a new antibiotic in medicine. Nat Prod Res 13(2):79–83Google Scholar
  19. Hultmark D (1998) Quantification of antimicrobial activity, using the inhibition-zone assay. Techniques in Insect Immunology 103–107Google Scholar
  20. Jenssen H, Hamill P, Hancock REW (2006) Peptide antimicrobial agents. Clin Microbiol Rev 19(3):491–511Google Scholar
  21. Kang SJ, Park SJ, Mishigochir T, Lee BJ (2014) Antimicrobial peptides: therapeutic potentials. Expert Rev Anti-Infect Ther 12(12):1477–1486Google Scholar
  22. Klockgether J, Tümmler B (2017) Recent advances in understanding Pseudomonas aeruginosa as a pathogen. F1000Res 6:1261Google Scholar
  23. Kruis W (2004) Review article: antibiotics and probiotics in inflammatory bowel disease. Aliment Pharmacol Ther 20(4):75–78Google Scholar
  24. Lee E, Shin A, Kim Y (2015) Anti-inflammatory activities of cecropin A and its mechanism of action. Arch Insect Biochem Physiol 88(1):31–44Google Scholar
  25. Maisch T (2007) Revitalized strategies against multi-resistant bacteria: antimicrobial photodynamic therapy and bacteriophage therapy. Anti Cancer Agents Med Chem 6(2):145–150Google Scholar
  26. Mishra B, Reiling S, Zarena D, Wang G (2017) Host defense antimicrobial peptides as antibiotics: design and application strategies. Curr Opin Chem Biol 38:87–96Google Scholar
  27. Monincova L, Budesinsky M, Slaninova J (1977) Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs. Insect Soc 24(1):9–36Google Scholar
  28. Mourgues F, Chevreau E, Brisset MN (1999) Antibacterial effect of cecropins on Erwinia amylovora pear cells interaction/a preliminary study. Acta Hortic 489(489):251–252Google Scholar
  29. Nan YH, Park KH, Park Y, Jeon YJ, Kim Y, Park IS, Hahm KS, Shin SY (2010) Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin. FEMS Microbiol Lett 292(1):134–140Google Scholar
  30. Papo N, Shai Y (2003) Can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes? Peptides 24(11):1693–1703Google Scholar
  31. Petrus EM, Chai S, Tunung A, Chai N, And LF, Son R (2011) A study on the minimum inhibitory concentration and minimum bactericidal concentration of nano colloidal silver on food-borne pathogens. Int Food Res J 18(18):55–66Google Scholar
  32. Prenner EJ, Lewis RN, Neuman KC, Gruner SM, Kondejewski LH, Hodges RS, Mcelhaney RN (1997) Nonlamellar phases induced by the interaction of gramicidin S with lipid bilayers. A possible relationship to membrane-disrupting activity. Biochemistry 36(25):7906–7916Google Scholar
  33. Rowshan HH, Keith K, Baur D, Skidmore P (2008) Pseudomonas aeruginosa infection of the auricular cartilage caused by “high ear piercing”: a case report and review of the literature. J Oral Maxillofac Surg 66(3):543–546Google Scholar
  34. Saikia K, Sravani YD, Ramakrishnan V, Chaudhary N (2017) Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB. Sci Rep 7:42994Google Scholar
  35. Scudiero O, Galdiero SM, Di NR, Vitiello M, Galdiero M, Naclerio G, Cassiman JJ, Pedone C, Castaldo G, Salvatore F (2010) Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity. Antimicrob Agents Chemother 54(6):2312–2322Google Scholar
  36. Silvestro L, Gupta KWeiser JN, Axelsen PH (1997) The concentration-dependent membrane activity of cecropin A. Biochemistry 36(38):11452–11460Google Scholar
  37. Silvestro L, Weiser JN, Axelsen PH (2000) Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob Agents Chemother 44(3):602–607Google Scholar
  38. Sudagidan M, Yemenicioğlu A (2012) Effects of nisin and lysozyme on growth inhibition and biofilm formation capacity of Staphylococcus aureus strains isolated from raw milk and cheese samples. J Food Prot 75(9):1627–1633Google Scholar
  39. Travkova OG, Moehwald H, Brezesinski G (2017) The interaction of antimicrobial peptides with membranes. Adv Colloid Interf Sci 247:521–532Google Scholar
  40. Tzialla C, Civardi E, Pozzi M, Stronati M (2015) Antibiotics and multi-resistant organisms. Ital J Pediatr 41(1):A45Google Scholar
  41. Liaoning Normal University, Jiang M (1993) Research on a kind of natural bio-preservatives—cecropins of Antheraea pernyi pupae. Food Sci.02Google Scholar
  42. Van den Ent F, Löwe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67(1):67–74Google Scholar
  43. Wachinger M, Kleinschmidt A, Winder D, Von Pechmann N, Ludvigsen A, Neumann M, Holle R, Salmons B, Erfle V, Brack-Werner R (1998) Antimicrobial peptides melittin and cecropin inhibit the replication of HIV-1 by suppressing viral gene expression. J Gen Virol 79(Pt 4):731–740Google Scholar
  44. 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):163–175Google Scholar
  45. Xia L, Liu Z, Ji M, Sun S, Yang J, Zhang F (2013) Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae. Protein Expr Purif 90(1):47–54Google Scholar
  46. Xing L, Wu W, Zhou B, Lin Z (2011) Streamlined protein expression and purification using cleavable self-aggregating tags. Microb Cell Factories 10(1):1–7Google Scholar
  47. Xu W, Zhao Q, Xing L, Lin Z (2016) Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag. Sci Rep 6.
  48. Yang YH, Fu SG, Peng H, Shen AD, Yue SJ, Go YF, Yuan L, Jiang ZF (1993) Abuse of antibiotics in China and its potential interference in determining the etiology of pediatric bacterial diseases. Pediatr Infect Dis J 12(12):986–988Google Scholar
  49. Yu H, Li H, Gao D, Gao C, Qi Q (2015) Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner. J Biotechnol 214:77–82Google Scholar
  50. Zhang M, Shan Y, Gao H, Wang B, Liu X, Dong Y, Liu X, Yao N, Zhou Y, Li X (2018) Expression of a recombinant hybrid antimicrobial peptide magainin II-cecropin B in the mycelium of the medicinal fungus Cordyceps militaris and its validation in mice. Microb Cell Factories 17(1):18Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhouChina
  2. 2.Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhouChina

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