重组环状胸腺五肽结构类似物Cyclo-(Cys-Arg-Lys-Asp-Val-Tyr-)的制备、鉴定和活性检测

Preparation, Characterization and Bioactivity Assay of a Novel Recombinant Tp-5 Cyclic Analogue Cyclo-(Cys-Arg-Lys-Asp-Val-Tyr-)

为了实现蛋白内含肽(Intein)介导的重组环状胸腺五肽结构类似物[cyclo-(Cys-Arg-Lys-Asp-Val-Tyr-),cTP]的高效制备,设计并合成编码6个氨基酸的cTP基因,克隆到表达载体pTWIN1,重组表达质粒pTW-cTp转化E.coliER2566构建工程菌,IPTG诱导由几丁质结合域纯化标签(chitinbindingdomain,CBD)、2个蛋白内含肽和目的多肽组成的“多元”融合蛋白(CBD-intein1-cTP-intein2-CBD)的高效表达.几丁质柱亲合层析纯化融合蛋白后,改变pH值和温度诱导intein1C端切割,硫醇MESNA诱导intein2N端切割,释放N端为Cys,C端为硫酯的重组cTP线性前体,通过非保护多肽硫酯环合法实现环肽生成.激光飞行质谱结果显示,纯化产物的分子量为764·4,与环肽的理论值相符.免疫活性检测结果显示,环肽cTP较线性多肽TP-5具有更显著的促进巨噬细胞吞噬能力的活性(P<0·01)和促进B细胞抗体生成的活性(P<0·01).

In order to obtain the recombinant Tp-5 cyclic analogue [cyclo-（Cys- Arg-Lys -Asp-Val-Tyr-）, cTP] efficiently by intein-mediated single column purification, a gene encoding 6-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector p TWIN1. The recombinant vector pTW-cTP was transferred into E. coli ER2566 cells. The multiplex fusion protein composed of two purification tags （chitin binding domain, CBD）, inteinl, intein2 and target peptide was expressed and purified by chintinaffinity chromatography. The C-terminal cleavage of inteinl was induced by changing the temperature and pH; and MESNA （2-mercaptoethanesulfonic acid ） was used to induce N-terminal cleavage of intein2. The precursors with an N-terminal cysteine and a C-terminal thioester was released and cyclized through the mechanism of macrocyclization of unprotected peptide precursors. SDS-PAGE assays indicated that the expression efficiency of soluble fusion protein reached 29 % of total soluble protein. The cTP was identified on Tricine-SDS-PAGE and the mass spectrometry assay showed that the cyclic peptide products contained the component with the molecular weight of 764.4 consistent with the theoretic value of cTP. Compared with the liner Tp-5, the recombinant cyclic peptide cTP displayed a stronger enhanced effect on the phagocytic function of macrophages and the formations of hemocytolysis blank in normal mice.