The antimicrobial peptide NZ17074, which is derived from arenicin-3 isolated from Arenicola marina, displayed high activity against a broad range of pathogenic bacteria and fungi. However, NZ17074 has not been produced using fermentation technology. The aim of this work was to study the expression of difficult-to-express NZ17074 in Pichia pastoris by fusing with SUMO3. The DNA fragments of NZ17074 and SUMO3 were fused into SUMO3-NZ17074 using overlap PCR and cloned into the pPICZαA vector to construct the pPICZ-SUMO3-NZ17074 expression vector. The rSUMO3-NZ17074 fusion protein, purified by Ni2+ -chelating affinity chromatography, was cleaved by 50% formic acid at 50°C for 28 h to release recombinant NZ17074 (rNZ17074). After purification with second affinity column, 4·1 mg rNZ17074 peptide with the purity over 90% was obtained from per litre fermentation culture. The rNZ17074 peptide exhibited the significant inhibition activity against Gram-negative bacteria: its minimal inhibitory concentrations (MICs) against Escherichia coli, Salmonella enteritidis and Pseudomonas aeruginosa were 2–4, 2 and 8–16 μg ml−1, respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide.Significance and Impact of the Study:
Recombinant active NZ17074 was produced with Pichia pastoris by using high-density fermentation technology for the first time. Our findings demonstrated the usefulness of SUMO-fusion technology as an effective expression strategy for synthesizing peptides in yeast. This SUMO3 expression system with a lower cost would likely be widely used for the production of other cytotoxic proteins including antimicrobial peptides.