黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

Expression and purification of an antimicrobial peptide, YFGAP from yellowfin tuna(Thunnus albacores) in Escherichia coli and characterization of its biological activity

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 k D,对革兰氏阳性菌(G+)和革兰氏阴性菌(G-)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体p ET22b-ELP20-EK-YFGAP、p ET22b-ELP40-EK-YFGAP和p ET22b-ELP40-L-EKYFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。

[Objective] The antimicrobial peptide, YFGAP from yellowfin tuna （Thunnus albacores）, consisted of 32 amino acid residues, shows broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria without hemolytic activity. Clone and express the antimicrobial peptides, YFGAP in Escherichia coli BL21（DE3） and characterize its bioactivity. [Methods] Based on the amino acids of EK-YFGAP and L-EK-YFGAP, the genes Were synthesized and ligated into pET-22b to construct these expression vectors, pET22b-ELP20-EK-YFGAP, pET22b-ELP40-EK-YFGAP and pET22b-ELP40-L-EK-YFGAP. The vectors were transformed into E. coli BL21（DE3） to express the fusion proteins. The fusion proteins were purified using the Elastin-like polypeptides （ELPs） as a purification tag by the method called inverse transition cycling （ITC） and digested by enterokinase. Then the recombinant YFGAP was purified using Vivaspin Turbo. Bioactivity of the peptide was tested. [Results] The fusion proteins, ELP40-EK-YFGAP and ELP40-L-EK-YFGAP were expressed and purified by two rounds of ITC. After digested by enterokinase, the recombinant YFGAP was purified using Vivaspin Turbo. Antimicrobial activity assay demonstrated the recombinant YFGAP exhibited high antibacterial activity against four fish pathogens without significant hemolytic activity. [Conelusion] The recombinant YFGAP was successfully expressed in E. coli using ELPs as a purification tag. This strategy does not require the use of chromatography, so that it is cost effective, easy to scale up and to multiplex. This study provides theoretical foundation and technical means for scale-up preparation of antimicrobial peptides by engineering method.