牛乳铁蛋白素(1-15)-蜂毒素(5-12)杂合肽基因的合成及在大肠杆菌中的表达

Cloning and expression of a gene encoding shortened LfcinB(1-15)-Melittin(5-12) hybrid peptide in Escherichia coli BL21(DE3)

摘要:

为了构建新型的杂合肽, 设计了一种新型杂合抗菌肽牛乳铁蛋白素(1-15)-蜂毒素(5-12), 由牛乳铁蛋白素(LfcinB)N端第1~15个氨基酸残基和蜂毒素(Melittin)N端第5~12个氨基酸残基组成。根据大肠杆菌密码子的偏爱性, 设计合成了杂合肽LfcinB(1-15)-Melittin(5-12)的基因片段, 插入到表达载体pET-32a的Nco I和Sal I的酶切位点之间, 构建重组表达质粒。重组表达质粒转化到Escherichia coli BL21(DE3)中, 经异丙基-b-D-硫代半乳糖苷(IPTG)诱导表达, 融合蛋白以可溶形式在Escherichia coli BL21(DE3)中获得成功表达, 表达量占菌体总蛋白的35%以上, 每升培养物可获得35 mg融合蛋白。带His标签的融合蛋白经His×Bind纯化试剂盒纯化、肠激酶切割和抑菌实验表明, 杂合肽具有明显的抑菌效果。这为利用基因工程方法生产抗菌肽奠定了理论基础。

Abstract:

In order to get new antibacterial peptide, we designed a hybrid peptide LfcinB(1-15)-Melittin(5-12), composed of 1-15 amino acid residues of bovine Lactoferricin and 5-12 amino acid residues of Melittin. According to the bias of codon utilization of Escherichia coli, We synthesized the gene encoding the hybrid peptide. We inserted the gene between the sites of Nco ? and Sal ? of pET-32a and obtained the recombinant expression vector for heterologous expression of LfcinB(1-15)-Melittin(5-12) in Escherichia coli. We used Escherichia coli BL21(DE3) as expression host for the recombinant plasmid. After induced by isopropyl-β-D-thiogalactoside (IPTG) under the optimized conditions, we realized the fusion protein was successfully expressed. The fusion protein was expressed in soluble form and the level was more than 35% of the total proteins. With (His)6×Tag, the fusion protein was easily purified by His×Bind Purification Kit. After purification, we obtained 35 mg of fusion protein from 1 L of culture medium. At last, we accomplished that the peptide LfcinB(1-15)-Melittin(5-12) was released from the fusion protein cleaved by enterokinase. The recombinant LfcinB(1-15)-Melittin(5-12) showed antimicrobial activity assayed by agar diffusion test. This is the first report on the heterologous expression of the hybrid antibacterial peptide LfcinB(1-15)-Melittin(5-12) in Escherichia coli and also provides basis for next cost-effective expression of other antimicrobial peptides in genetic engineering.