Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner. (2nd April 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: 2014-04-02
• Subjects: Microbiology -- Periodicals; 660.62
• DOI: 10.1111/lam.12246
• Languages: English
• ISSNs: 0266-8254

Abstract

<abstract abstract-type="main" id="lam12246-abs-0001"> <title>Abstract</title> <sec id="lam12246-sec-1001" sec-type="section"> <p>The antimicrobial peptide NZ17074, which is derived from arenicin‐3 isolated from <italic>Arenicola marina</italic>, 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 <italic>Pichia pastoris</italic> by fusing with SUMO3. The DNA fragments of NZ17074 and SUMO3 were fused into SUMO3‐NZ17074 using overlap PCR and cloned into the pPICZ<italic>α</italic>A vector to construct the pPICZ‐SUMO3‐NZ17074 expression vector. The rSUMO3‐NZ17074 fusion protein, purified by Ni<sup>2</sup> <sup>+</sup> ‐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 <italic>Escherichia coli</italic>, <italic> Salmonella enteritidis</italic> and <italic>Pseudomonas aeruginosa</italic> were 2–4, 2 and 8–16 <italic>μ</italic>g ml<sup>−1</sup>, respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide.</p> </sec> <sec id="lam12246-sec-1002" sec-type="section"> <title>Significance and Impact of the Study</title> <p>Recombinant active NZ17074 was produced with <italic>Pichia pastoris</italic> 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.</p> </sec> </abstract>