Harnessing the Power of GFP Fusion in E. coli for Well-Optimized Expression and Enhanced Characterization of Human Hepcidin-25

Antimicrobial peptides (AMPs) are natural compounds with broad-spectrum activity, playing a key role in the innate immune system by disrupting pathogen membranes. This study evaluates the recombinant antimicrobial peptide GFP-Hepc25, where GFP tagging facilitates fluorescence monitoring without additional staining. Optimal growth conditions for GFP-Hepc25 expression were determined as 18°C, IPTG concentrations of 0.1–1 mM, and a casein-based medium with yeast extract and NaCl. Both GFP-Hepc25 and Hepc25 demonstrated antimicrobial activity against E. coli and S. aureus . GFP-Hepc25 exhibited notable stability under heat, and acidic conditions, as well as in the presence of MnCl₂, ZnSO₄, MgSO₄, CuSO₄, and FeSO₄. It retained stability at 100°C for 5 and 10 minutes, though prolonged heating caused degradation. However, stability decreased under alkaline pH and with detergents such as SDS, Triton-X100, and Tween20. GFP-Hepc25 significantly inhibited S. aureus and P. aeruginosa biofilm formation. These findings highlight GFP-Hepc25 as a promising next-generation antimicrobial peptide.