Recombinant expression of a novel Mytilus defensin in Pichia pastoris

Mytilus contain abundant antimicrobial peptides (AMPs) that play a key role in the innate immunity. However, heterologous production of these AMPs remains challenging due to their short sequences, multiple disulfide bonds, and high content of cationic amino acids, which hinder functional expression in prokaryotic systems such as Escherichiacoli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies, we reported the successful recombinant expression of myticofensin-B1, a novel defensin-like AMP identified previously in Mytiluscoruscus, using the eukaryotic host Pichiapastoris. The codon-optimized gene encoding the mature myticofensin-B1 (composed of 65 amino acid residues, including 6 conserved cysteine residues) was cloned into a pPICZαA vector and expressed in P. pastoris GS115. Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), showing a molecular weight of 8 849.9 Da, which was consistent with the theoretical prediction. Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1, with stronger inhibition against Gram-negative bacteria. Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria. In addition, the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells. This study establishes P. pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.