Design and chromatography-free purification of a recombinant helix-bundle AMP with potent antibacterial activity for pork preservation

The potential of antimicrobial peptides as food preservatives is being increasingly recognized, yet several issues remain to be addressed, including instability, toxicity, and high production costs. In this study, we engineered a chimeric fusion of the AMP PR-FO (a hybrid peptide composed of porcine PMAP-36 and avian Fowlicidin-2 variants) and a hyperstable four-helix bundle domain (DAMP4), which was expressed in Pichia pastoris . Leveraging the intrinsic thermostability of the helix bundle, we developed a selective thermal precipitation based chromatographic-free purification process, yielding 79.02 mg/L with 85.19% purity, while preserving the antimicrobial potency of PR-FO. The recombinant peptide, rD2L exhibited potent broad-spectrum antibacterial activity (GM MIC = 2.31 μmol/L). Notably, rD2L demonstrated exceptional tolerance to proteases, heat, and physiological salts, maintaining a favorable biosafety profile. Mechanistic studies revealed that rD2L primarily acted through membrane depolarization and disruption, with supplementary DNA-binding activity, significantly reducing the development of resistance. Furthermore, rD2L effectively inhibited bacterial proliferation and suppressed lipid oxidation, thereby delaying spoilage of chilled pork over a 5-day storage period. These findings demonstrate that the platform we developed integrates cost-effective biomanufacturing with functional robustness and safety, making it a promising candidate for industrial food preservation.