Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner. (1st July 2014)

• Record Type: Journal Article
• Authors: Wang, X.J.; Wang, X.M.; Teng, D.; Zhang, Y.; Mao, R.Y.; Wang, J.H.
• Is Part Of: Letters in applied microbiology. Volume 59:Number 1(2014:Jul.)
• Page Start: 71
• Page End: 78
• Publication Date: 2014-07-01
• Subjects: antimicrobial activity -- antimicrobial peptide -- NZ17074 -- Pichia pastoris -- recombinant expression -- SUMO3; Microbiology -- Periodicals; 660.62
• DOI: 10.1111/lam.12246
• Languages: English
• ISSNs: 0266-8254

Abstract

Abstract: 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 pPICZ α A vector to construct the pPICZ‐SUMO3‐NZ17074 expression vector. The rSUMO3‐NZ17074 fusion protein, purified by Ni 2 + ‐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 μ g 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.