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Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches

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Abstract

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni–NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

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Acknowledgements

This study has been written by the authors.

Funding

The study was financially supported by Fasa University of Medical Sciences (Project Number:99094) and was approved by the Ethical Committee of Fasa University of Medical Sciences (IR.FUMS.REC.1399.137).

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Authors and Affiliations

  1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Fasa University of Medical Sciences, Fasa, Iran

    Mahsa Abbasi & Shirin Mahmoodi

  2. School of Advanced Technologies in Medicine, Fasa University of Medical Sciences, Fasa, Iran

    Esmail Behmard

  3. Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

    Mohammad Hashem Yousefi & Seyed Shahram Shekarforoush

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  1. Mahsa Abbasi
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  2. Esmail Behmard
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  3. Mohammad Hashem Yousefi
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  4. Seyed Shahram Shekarforoush
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  5. Shirin Mahmoodi
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Contributions

MA and SHM conceived and designed the research. MA, SHM, MHY, and SSHSH conducted experiments. MA, EB contributed analytical tools. All authors wrote the manuscript and approved it.

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Correspondence to Shirin Mahmoodi.

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Communicated by Yusuf Akhter.

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Abbasi, M., Behmard, E., Yousefi, M.H. et al. Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches. Arch Microbiol 205, 199 (2023). https://doi.org/10.1007/s00203-023-03529-5

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