Key Role of Disulfide Bridges in the Antimicrobial Activity of Beta-Defensin from Olive Flounder
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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.
KeywordsBeta-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).
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Conflict of interest
The authors have no potential conflict of interest to declare.
- 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
- 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
- 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