Key Role of Disulfide Bridges in the Antimicrobial Activity of Beta-Defensin from Olive Flounder

  • Yunqi Ma
  • So-Sun Kim
  • Chang-Hyun Maeng
  • David Nahm-Joon Kim
  • Chang-Joo Lee
  • Bo-Hye Nam
  • Youg-Ok Kim
  • Cheul-Min An
  • Jang-Su ParkEmail author


Proteins that contain multiple disulfide bonds (SS bonds) expressed in Escherichia coli are usually problematic. This study reports the successful recombinant expression of the antimicrobial peptide β-defensin isolated from olive flounder in E. coli. The native form of β-defensin contained three discrete disulfide bridges: Cys1–Cys5, Cys2–Cys4, Cys3–Cys6. We constructed a periplasmic expression vector using small leading transmembrane protein YoaJ, and eventually, isolated bioactive β-defensin, which was then subjected to mass spectroscopy, circular dichroism spectroscopy, and anti-microbial testing. Results indicated bioactive β-defensin with a properly folded and native structure was formed. To investigate the roles of SS bonds, site-directed mutation method was applied to disrupt one, two, or three disulfide bridges. A dose-dependent effect was observed when more disulfide bridges were broken and a correlation between structure and function was observed, which further illustrated the key roles of SS bonds in maintaining the conserved motif and secondary structure of olive flounder beta-defensin.


Beta-defensin Antimicrobial activity Disulfide bond Olive flounder 



This research was supported by grants from the Marine Biotechnology Program (Grant No. 20170305; Development of Biomedical materials based on marine proteins) Funded by the Korean Ministry of Oceans and Fisheries, and from the National Fisheries Research and Development Institute, Korea (R2019016).

Compliance with Ethical Standards

Conflict of interest

The authors have no potential conflict of interest to declare.


  1. Daiho T, Yamasaki K, Saino T, Kamidochi M, Satoh K, Iizuka H, Suzuki H (2001) Mutations of either or both Cys876 and Cys888 residues of sarcoplasmic reticulum Ca2+-ATPase result in a complete loss of Ca2+ transport activity without a loss of Ca2+-dependent ATPase activity. Role of the CYS876-CYS888 disulfide bond. J Biol Chem 276(35):32771–32778CrossRefGoogle Scholar
  2. Dhople V, Krukemeyer A, Ramamoorthy A (2006) The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochim Biophys Acta 1758(9):1499–1512CrossRefGoogle Scholar
  3. Fang X, Peng L, Xu Z, Wu J, Cen P (2002) Cloning and expression of human beta-defensin-2 gene in Escherichia coli. Protein Pept Lett 9(1):31–37CrossRefGoogle Scholar
  4. Hatahet F, Nguyen VD, Salo KE, Ruddock LW (2010) Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microb Cell Fact 9:67Google Scholar
  5. Humphreys DP, Chapman AP, Reeks DG, Lang V, Stephens PE (1997) Formation of dimeric Fabs in Escherichia coli: effect of hinge size and isotype, presence of interchain disulphide bond, Fab’ expression levels, tail piece sequences and growth conditions. J Immunol Methods 209(2):193–202CrossRefGoogle Scholar
  6. Ivanenkov VV, Murphy-Piedmonte DM, Kirley TL (2003) Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 (CD39L2) nucleotidase: implications for structure and function. Biochemistry 42(40):11726–11735CrossRefGoogle Scholar
  7. Jain A, Yadav BK, Chugh A (2015) Marine antimicrobial peptide tachyplesin as an efficient nanocarrier for macromolecule delivery in plant and mammalian cells. FEBS J 282(4):732–745CrossRefGoogle Scholar
  8. Jin JY, Zhou L, Wang Y, Li Z, Zhao JG, Zhang QY, Gui JF (2010) Antibacterial and antiviral roles of a fish beta-defensin expressed both in pituitary and testis. PLoS ONE 5(12):e12883CrossRefGoogle Scholar
  9. Katzen F, Beckwith J (2002) Disulfide bond formation in periplasm of Escherichia coli. Methods Enzymol 348:54–66CrossRefGoogle Scholar
  10. Lacerda AF, Vasconcelos EA, Pelegrini PB, Grossi de Sa MF (2014) Antifungal defensins and their role in plant defense. Front Microbiol 5:116CrossRefGoogle Scholar
  11. Latiffi AA, Salleh AB, Rahman RN, Oslan SN, Basri M (2013) Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and alpha-factor secretion signal in Pichia pastoris. Genes Genet Syst 88(2):85–91CrossRefGoogle Scholar
  12. Li CL, Zhao YC, Song XY, Huang XX, Zhao WD (2013) Molecular cloning, expression and characterization of the porcine beta defensin 2 in E. coli. Protein Pept Lett 20(6):715–723CrossRefGoogle Scholar
  13. Malhotra S, Silo-Suh LA, Mathee K, Ohman DE (2000) Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of the disulfide bond isomerase, DsbA. J Bacteriol 182(24):6999–7006CrossRefGoogle Scholar
  14. Montero-Alejo V, Acosta-Alba J, Perdomo-Morales R, Perera E, Hernandez-Rodriguez EW, Estrada MP, Porto-Verdecia M (2012) Defensin like peptide from Panulirus argus relates structurally with beta defensin from vertebrates. Fish Shellfish Immunol 33(4):872–879CrossRefGoogle Scholar
  15. Nam BH, Moon JY, Kim YO, Kong HJ, Kim WJ, Lee SJ, Kim KK (2010) Multiple beta-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus. Fish Shellfish Immunol 28(2):267–274CrossRefGoogle Scholar
  16. Ricq EL, Hooker JM, Haggarty SJ (2016) Activity-dependent regulation of histone lysine demethylase KDM1A by a putative thiol/disulfide switch. J Biol Chem 291(47):24756–24767CrossRefGoogle Scholar
  17. Ruangsri J, Kitani Y, Kiron V, Lokesh J, Brinchmann MF, Karlsen BO, Fernandes JM (2013) A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity. PLoS ONE 8(4):e62302CrossRefGoogle Scholar
  18. Skube SB, Chaverri JM, Goodson HV (2010) Effect of GFP tags on the localization of EB1 and EB1 fragments in vivo. Cytoskeleton (Hoboken) 67(1):1–12CrossRefGoogle Scholar
  19. Stotz HU, Thomson JG, Wang Y (2009) Plant defensins: defense, development and application. Plant Signal Behav 4(11):1010–1012CrossRefGoogle Scholar
  20. Sung DK, Chang YS, Sung SI, Yoo HS, Ahn SY, Park WS (2016) Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta-defensin-2 via toll-like receptor 4 signalling. Cell Microbiol 18(3):424–436CrossRefGoogle Scholar
  21. Terras FR, Penninckx IA, Goderis IJ, Broekaert WF (1998) Evidence that the role of plant defensins in radish defense responses is independent of salicylic acid. Planta 206(1):117–124CrossRefGoogle Scholar
  22. Thevissen K, Ferket KK, Francois IE, Cammue BP (2003) Interactions of antifungal plant defensins with fungal membrane components. Peptides 24(11):1705–1712CrossRefGoogle Scholar
  23. van den Hengel LG, Osanto S, Reitsma PH, Versteeg HH (2013) Murine tissue factor coagulant activity is critically dependent on the presence of an intact allosteric disulfide. Haematologica 98(1):153–158CrossRefGoogle Scholar
  24. Zhang L, Chou CP, Moo-Young M (2011) Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system. Biotechnol Adv 29(6):923–929CrossRefGoogle Scholar
  25. Zhu ZC, Gupta KK, Slabbekoorn AR, Paulson BA, Folker ES, Goodson HV (2009) Interactions between EB1 and microtubules: dramatic effect of affinity tags and evidence for cooperative behavior. J Biol Chem 284(47):32651–32661CrossRefGoogle Scholar
  26. Zou J, Mercier C, Koussounadis A, Secombes C (2007) Discovery of multiple beta-defensin like homologues in teleost fish. Mol Immunol 44(4):638–647CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019
corrected publication 2019

Authors and Affiliations

  1. 1.Department of Chemistry and Chemistry Institute of Functional MaterialsPusan National UniversityBusanRepublic of Korea
  2. 2.Biotechnology Research Division, Aquaculture Industry DepartmentNational Institute of Fisheries ScienceBusanRepublic of Korea

Personalised recommendations