Design and Recombinant Production of Proline-rich Antimicrobial Peptide 385P

Abstract The rapid emergence of antimicrobial resistance has increased the search for alternative agents with novel mechanisms of action. Here, we established an end-to-end framework for the rational development of proline-rich antimicrobial peptides (PrAMPs), integrating computational motif discovery, peptide design, recombinant production, and biological characterization. Conserved motifs from highly active PrAMPs were identified and ranked using MEME and rationally assembled to generate the 36-amino acid peptide 385P. A C-terminal hexahistidine-tagged construct (r385P) was successfully produced in Pichia pastoris GS115 and confirmed by SDS-PAGE, Western blotting, and His-tag ELISA. r385P exhibited broad-spectrum antimicrobial activity, with MICs of 4–32 µg/mL against Gram-negative bacteria, 2–32 µg/mL against Gram-positive bacteria, and 64 µg/mL against Candida albicans and Candida glabrata , with the highest antibacterial activity against Listeria monocytogenes and Acinetobacter baumannii . r385P exhibited strong antibiofilm activity, achieving 83.83% inhibition against Escherichia coli O157:H7 at MIC/2 and complete inhibition against Staphylococcus aureus at MIC. FICI assays revealed antibiotic- and species-dependent interactions, including synergy with kanamycin against Klebsiella pneumoniae (FICI = 0.375) and consistent additive interactions with tetracycline. r385P retained full antimicrobial activity after exposure to 50–200 mM NaCl and 1–4 h of trypsin treatment, while MICs increased two- and four-fold following exposure to 65 and 95°C, respectively. Hemolysis remained below 4% at 256 µg/mL, while MRC-5 cell viability was 71% at 320 µg/mL. Collectively, these findings demonstrate the potential of conserved-motif assembly as a practical strategy for developing functionally robust PrAMP candidates.