Abstract
A recombinant version of the AGAAN antimicrobial peptide (rAGAAN) was cloned, expressed, and purified in this study. Its antibacterial potency and stability in harsh environments were thoroughly investigated. A 15 kDa soluble rAGAAN was effectively expressed in E. coli. The purified rAGAAN exhibited a broad antibacterial spectrum and was efficacious against seven Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration (MIC) of rAGAAN against the growth of M. luteus (TISTR 745) was as low as 60 µg/ml. Membrane permeation assay reveals that the integrity of the bacterial envelope is compromised. In addition, rAGAAN was resistant to temperature shock and maintained a high degree of stability throughout a reasonably extensive pH range. The bactericidal activity of rAGAAN ranged from 36.26 to 79.22% in the presence of pepsin and Bacillus proteases. Lower bile salt concentrations had no significant effect on the function of the peptide, whereas higher concentrations induced E. coli resistance. Additionally, rAGAAN exhibited minimal hemolytic activity against red blood cells. This study indicated that rAGAAN may be produced on a large scale in E. coli and that it had an excellent antibacterial activity and sufficient stability. This first work to express biologically active rAGAAN in E. coli yielded 8.01 mg/ml at 16 °C/150 rpm for 18 h in Luria Bertani (LB) medium supplemented with 1% glucose and induced with 0.5 mM IPTG. It also assesses the interfering factors that influence the activity of the peptide, demonstrating its potential for research and therapy of multidrug-resistant bacterial infections.
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All the data has been presented in the paper and any additional information will be available on request.
Abbreviations
- MIC:
-
Minimum Inhibitory Concentration
- PI:
-
Propidium Iodide
- MHB:
-
Mueller Hinton Broth
- IPTG:
-
Isopropyl-1-thio-β-D-galactopyranoside
- LC–MS/MS:
-
Liquid chromatography-tandem mass spectrometry
- SDS:
-
PAGE – Sodium dodecyl Sulphate–Polyacrylamide Gel Electrophoresis
- KDa:
-
Kilo Dalton
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Acknowledgements
This research was supported by King Mongkut’s University of Technology Thonburi’s Postdoctoral Fellowship.
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Funding from Research, Innovation and Partnerships Office, King Mongkut’s University of Technology Thonburi, KMUTT.
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YSA performed the experiments and original draft of the manuscript. NR assisted in performing the experiments. NUJ proofread the manuscript and provide technical writing assistance. YK data curation, conceptualization, and discussion. NJ funding acquisition, methodology, data curation and supervision.
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Ajingi, Y.S., Rukying, N., Jiddah, N.U. et al. Cloning, recombinant expression, purification, and functional characterization of AGAAN antibacterial peptide. 3 Biotech 13, 88 (2023). https://doi.org/10.1007/s13205-023-03512-3
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DOI: https://doi.org/10.1007/s13205-023-03512-3