ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 9, pp. 1610-1618 © Pleiades Publishing, Ltd., 2024.
1610
Efficient Production and Purification
of Bioactive E50-52-Class IIa 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-spec-
trum 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 recombi-
nant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-class III bacteriocin (LssCAT),
which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-ex-
pression in Escherichia coli, but it became soluble using phosphate buffer (pH 7.4) supplemented with 8 M urea.
Purification using immobilized-Ni 2+ affinity chromatography under urea denaturing conditions resulted in con-
sistent 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-de-
pendent 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 100 mg per liter of culture.
To the best 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.