Wnt-1信号通路靶蛋白WISP-1的原核表达与纯化

Protein expression and purification of Wnt-1 inducible-signaling pathway protein 1 （WISP-1）

目的获得纯度较高的WISP-1蛋白结构域，为进一步开发靶向WISP-1蛋白的药物奠定基础。方法将编码WISP-1蛋白4个结构域的基因克隆到pFN19A蛋白表达载体中，4个重组质粒分别在大肠埃希菌中诱导表达对应的WISP-1蛋白结构域。用Halo树脂共价层析法纯化目的蛋白。结果获得了4个重组质粒pFNl9AHalo-IGFBP／VWFC／TSP／CTCK。并在KRX敏感菌株中诱导表达目的蛋白，通过优化诱导条件获得4个高表达结构域蛋白。SDS-PAGE和Western印迹特异性条带鉴定纯化蛋白的质量大小与预期一致。经BCA蛋白浓度监测，E2中重组蛋白浓度可达到500—1500mg／L。结论通过Halo树脂共价层析法，获得了纯度较高的WISP-14个结构域蛋白，为研究WISP-1蛋白在肝癌等肿瘤发生发展过程中的关键功能结构域打下基础。

Objective To achieve the pure recombinant proteins of WISP-1 domains and lay a foundation of fur- ther study on the targeting drug of protein WISP-1. Methods The genes which encoded four domains of WISP-1 protein with a halo tag at N-terminus （pFN19A vector）were cloned and the corresponding proteins were expressed in E. coli. The recombinant proteins were covalently immobilized onto HaloLink resins and purified by enzyme cleavage. Results The recombinant plasmids, namely pFN19A Halo-IGFBP, Halo-VWFC, Halo-TSP, Halo-CTCK, were transformed into the KRX competent cells to express proteins respectively. The induction conditions were optimized for high expression of the target proteins. Purified domain proteins were validated by SDS-PAGE and Western-blot, the results showed that purified domains had the expected molecular weights. The concentration of recombinant protein in E2 could reach 500-1500 mg/L by BCA protein concentration monitoring. Conclusions Four domains of WISP-1 protein are successfully purified by chromatography. These proteins are very important to better understand the key functional domains of WISP-1 in the de- velopment of the malignant tumors, such as hepatocellular carcinoma.