title: Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner.
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 pPICZαA 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 μg ml(-1) , respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: X J
authors.initials: XJ
authors.lastname: Wang
authors.forename: X M
authors.initials: XM
authors.lastname: Wang
authors.forename: D
authors.initials: D
authors.lastname: Teng
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: R Y
authors.initials: RY
authors.lastname: Mao
authors.forename: J H
authors.initials: JH
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects; D005285:Fermentation; D006090:Gram-Negative Bacteria / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010843:Pichia; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012477:Salmonella enteritidis / Q000187:drug effects; D014452:Ubiquitins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C000592841:NZ17074; D011993:Recombinant Fusion Proteins; C492189:SUMO3 protein, human; D014452:Ubiquitins
keywords: NZ17074; Pichia pastoris; SUMO3; antimicrobial activity; antimicrobial peptide; recombinant expression