Wang X J, Wang X M, Teng D, Zhang Y, Mao R Y, Wang J H
Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
Lett Appl Microbiol. 2014 Jul;59(1):71-8. doi: 10.1111/lam.12246. Epub 2014 Apr 2.
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.
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.
抗菌肽NZ17074源自从沙蠋中分离出的沙蠋素-3，对多种病原菌和真菌具有高活性。然而，NZ17074尚未采用发酵技术生产。本研究的目的是通过与SUMO3融合，研究难表达的NZ17074在毕赤酵母中的表达。利用重叠PCR将NZ17074和SUMO3的DNA片段融合成SUMO3-NZ17074，并克隆到pPICZαA载体中构建pPICZ-SUMO3-NZ17074表达载体。通过Ni(2)+螯合亲和层析纯化的rSUMO3-NZ17074融合蛋白，在50℃下用50%甲酸切割28小时以释放重组NZ17074（rNZ17074）。用第二个亲和柱纯化后，每升发酵培养物获得4.1mg纯度超过90%的rNZ17074肽。rNZ17074肽对革兰氏阴性菌表现出显著的抑制活性：其对大肠杆菌、肠炎沙门氏菌和铜绿假单胞菌的最小抑菌浓度（MIC）分别为2-4、2和8-16μg/ml，这表明SUMO3是有毒肽表达的良好融合伴侣。
首次利用毕赤酵母通过高密度发酵技术生产重组活性NZ17074。我们的研究结果证明了SUMO融合技术作为在酵母中合成肽的有效表达策略的实用性。这种成本较低的SUMO3表达系统可能会广泛用于生产包括抗菌肽在内的其他细胞毒性蛋白。
