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Recombinant production of the antimicrobial peptide NZ 17074 in P ichia pastoris using SUMO 3 as a fusion partner
Author(s) -
Wang X.J.,
Wang X.M.,
Teng D.,
Zhang Y.,
Mao R.Y.,
Wang J.H.
Publication year - 2014
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12246
Subject(s) - pichia pastoris , biology , recombinant dna , peptide , escherichia coli , fusion protein , biochemistry , antimicrobial peptides , microbiology and biotechnology , gene
The antimicrobial peptide NZ 17074, which is derived from arenicin‐3 isolated from A renicola marina , displayed high activity against a broad range of pathogenic bacteria and fungi. However, NZ 17074 has not been produced using fermentation technology. The aim of this work was to study the expression of difficult‐to‐express NZ 17074 in P ichia pastoris by fusing with SUMO 3. The DNA fragments of NZ 17074 and SUMO 3 were fused into SUMO 3‐ NZ 17074 using overlap PCR and cloned into the p PICZ α A vector to construct the p PICZ ‐ SUMO 3‐ NZ 17074 expression vector. The r SUMO 3‐ NZ 17074 fusion protein, purified by N i 2 + ‐chelating affinity chromatography, was cleaved by 50% formic acid at 50°C for 28 h to release recombinant NZ 17074 (r NZ 17074). After purification with second affinity column, 4·1 mg r NZ 17074 peptide with the purity over 90% was obtained from per litre fermentation culture. The r NZ 17074 peptide exhibited the significant inhibition activity against Gram‐negative bacteria: its minimal inhibitory concentrations ( MIC s) against E scherichia coli , S almonella enteritidis and Pseudomonas aeruginosa were 2–4, 2 and 8–16 μ g ml −1 , respectively, which indicated that SUMO 3 is a good fusion partner for the expression of the toxic peptide. Significance and Impact of the Study Recombinant active NZ 17074 was produced with P ichia 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 SUMO 3 expression system with a lower cost would likely be widely used for the production of other cytotoxic proteins including antimicrobial peptides.