LAUSR

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.