抗菌抗癌肽CM4N在大肠杆菌中的融合表达与活性鉴定

Fusion Expression of Antibacterial and Antitumorial Peptide CM4N in Escherichia coli and Its Activity Identification

为探讨内含肽intein在抗菌肽表达与纯化中的应用,采用递归PCR法合成CM4末端添加Asn的基因CM4N,克隆至E.coli表达载体pTYB11中,构建了与intein的C端融合的表达载体pTYB11-CM4N。重组质粒转化至E.coliBL21(DE3)中进行IPTG诱导表达,融合蛋白intein-CM4N主要以可溶形式存在于胞内。利用载体中intein中的chitin结合域,将融合蛋白通过chitin亲和层析一步纯化,经DTT诱导intein的自我切割活性,实现CM4N在亲和柱上的切割与分离,透析冻干后得到了纯度95%以上的重组CM4N。活性检测结果显示,重组CM4N具有抗大肠杆菌、沙门氏菌和K562肿瘤细胞的活性。研究认为,内含肽在抗菌肽的基因工程中可能具有重要的应用前景。

In order to obtain the recombinant antibacterial peptide CM4N efficiently, an intein mediated single column purification was used. The CM4N gene, which encodes 36 amino acids peptide with Asn at the C terminus, was synthesized by a recursive polymerase chain reaction strategy and cloned into Escherichia coli expression vector pTYBll. The recombinant vector pTYBll-CM4N was transferred in E. coli BL21 （DE3） cells and the peptide CM4N was expressed mainly in soluble form in fusion with an N-terminal intein tag. Then the fusion protein was purified by chitin-affinity chromatography and the self-cleavage activity of the intein was induced by 1,4-dithiothreitol. After dialyzation and freeze drying, the purity of recombinant CM4 was about 95%. The recombinant CM4N exhibited obvious activities a gainst the tested bacteria and K562 tumor cells. These results showed that the intein-mediated expression and purification system could have important application prospect in the production of recombinant antibacterial peptides.