Evaluation of a cathelicidin derivative fusion polypeptide for pathogen-targeted antimicrobial approach

Abstract The human cathelicidin LL-37 is one of the most promising candidates for replacing standard antibiotics due to its broad-spectrum activity and low propensity for resistance development. However, the widespread clinical adoption of antimicrobial peptides (AMPs) is currently limited by the high cost and several constraints of traditional chemical synthesis. Recombinant expression offers a cost-effective and scalable alternative, although challenges including host toxicity and low expression yields remain. In previous studies, the human elastin-like polypeptide (HELP) carrier effectively mitigated host toxicity, enhanced protein solubility, and enabled simple and convenient purification of antimicrobial domains through its thermo-responsive phase-transition properties. Here, we report the recombinant production of H1FK16, a non-cytotoxic polypeptide in which FK16, a bioactive LL-37-derived peptide, is genetically fused to the HELP carrier. Its production yield was comparable to that of the carrier, with an average of 180 mg of fusion protein per liter of bacterial culture. The construct was designed to retain an upstream glutamic acid residue that enables site-specific FK16 release by glutamyl endopeptidase, a proteolytic activity associated with Staphylococcus aureus virulence. The released FK16 domain retained its antimicrobial activity against both Gram-positive S. aureus and Gram-negative Escherichia coli , exhibiting a minimum inhibitory concentration of 2.5 µM, comparable to reported values. The findings highlight the potential of this fusion system as a versatile platform for developing biocompatible materials that selectively target pathogenic microorganisms through enzymatically triggered release of antimicrobial domains. Key points • The LL- 37-derived antimicrobial peptide FK16 was produced by recombinant fusion technology. • The active FK16 domain was released through an enzyme-triggered cleavage process. • H1FK16 treatment reduced biofilm biomass.