| Autor: |
Phrutpoom N; Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Nakornpathom, 73170, Thailand., Khaokhiew T; Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Nakornpathom, 73170, Thailand. tararat.kha@mahidol.ac.th., Linn AK; Excellent Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand., Sakdee S; Center for Advanced Therapeutics, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand., Imtong C; Bacterial Toxin Research Innovation Cluster, Biophysics Institute for Research and Development (BIRD), Chiang Mai, 50230, Thailand., Jongruja N; Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi (KMUTT), Bangkok, 10140, Thailand., Angsuthanasombat C; Center for Advanced Therapeutics, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand. chanan.ang@mahidol.ac.th.; Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand. |
| Abstrakt: |
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-Ni 2+ 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. |
Full text from SpringerLink