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
Issued Date
2024-09-01
Resource Type
ISSN
00062979
eISSN
16083040
Scopus ID
2-s2.0-85206250159
Journal Title
Biochemistry (Moscow)
Volume
89
Issue
9
Start Page
1610
End Page
1618
Rights Holder(s)
SCOPUS
Bibliographic Citation
Biochemistry (Moscow) Vol.89 No.9 (2024) , 1610-1618
Suggested Citation
Phrutpoom N., Khaokhiew T., Linn A.K., Sakdee S., Imtong C., Jongruja N., Angsuthanasombat C. 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. Biochemistry (Moscow) Vol.89 No.9 (2024) , 1610-1618. 1618. doi:10.1134/S0006297924090074 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/101677
Title
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
Corresponding Author(s)
Other Contributor(s)
Abstract
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-class III 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 (pH 7.4) supplemented with 8 M 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 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.