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

Abstract

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.