Cloning, recombinant expression, purification, and functional characterization of AGAAN antibacterial peptide

Ya'u Sabo Ajingi; Neeranuch Rukying; Nafiu Usman Jiddah; Yuichi Koga; Nujarin Jongruja

doi:10.1007/s13205-023-03512-3

Cloning, recombinant expression, purification, and functional characterization of AGAAN antibacterial peptide

3 Biotech. 2023 Mar;13(3):88. doi: 10.1007/s13205-023-03512-3. Epub 2023 Feb 18.

Authors

Ya'u Sabo Ajingi^{1

2}, Neeranuch Rukying¹, Nafiu Usman Jiddah^{1

3}, Yuichi Koga⁴, Nujarin Jongruja¹

Affiliations

¹ Department of Microbiology, Faculty of Science, King Mongkut's University of Technology (KMUTT), 126 Pracha Uthit Rd., Bang Mod, Thung Khru, Thonburi , 10140 Bangkok Thailand.
² Department of Biology, Faculty of Science, Kano University of Science and Technology (KUST), Wudil, Nigeria.
³ Department of Biochemistry, Faculty of Science, Gombe State University, Gombe, Nigeria.
⁴ Department of Applied Chemistry, Faculty of Engineering, Okayama University of Science , Ridai-cho 1-1, Kita-ku, Okayama City, Okayama Japan.

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.

Keywords: Antibacterial peptide; Propidium iodide; Purification; Recombinant expression; Stability.

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