Efficient Production and Purification of Bioactive E50-52-ClassIIa Peptidic Bacteriocin Is Achieved through Fusion with the Catalytic Domain of Lysostaphin-Class III Bacteriocin.

Abstract

E50-52, a class IIa-peptidic bacteriocin produced by a strain of Enterococcus faecium, has broad-spectrum antimicrobial activity against various foodborne pathogens. However, effective utilization of the E50-52 has been limited by low production yields and challenges associated with separation and purification of this 39-amino acid antimicrobial peptide. In this study, we have successfully produced a biologically active recombinant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-classIII bacteriocin (LssCAT), which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-expression in Escherichia coli, but it became soluble using phosphate buffer (pH7.4) supplemented with 8M urea. Purification using immobilized-Ni2+ affinity chromatography under urea denaturing conditions resulted in consistent production a homogenous products (LssCAT-E50-52) with >95% purity. The purified protein was refolded using an optimized stepwise dialysis process. The resulting refolded LssCAT-E50-52 protein exhibited dose-dependent inhibitory activity against Helicobacter pylori, a Gram-negative, flagellated, helical bacterium that is associated with gastric cancer. Overall, the optimized protocol described in this study effectively produced large quantities of high-purity recombinant LssCAT-E50-52 protein, yielding approximately 100mg per liter of culture. To thebest of our knowledge, this is the first report on the impact of LssCAT-E50-52 on H.pylori. This finding could pave the way for further research into bactericidal mechanism and potential applications of this bacteriocin in biomedical industry.