用硫氧还蛋白融合表达系统表达小鼠内皮抑素

Expression of mouse endostatin by using thioredoxin fusion expression system

目的利用硫氧还蛋白融合表达系统表达小鼠内皮抑素(endostatin)。方法采用PCR技术从pMFGendo质粒上扩增出小鼠endostatin完整的cDNA序列,将其克隆至硫氧还蛋白融合表达载体pThiohisA中,转化大肠杆菌BL21,经IPTG(异丙基-β-D-硫代半乳糖苷)诱导表达重组融合蛋白,SDS-PAGE分析表达产物为包涵体,将包涵体纯化后通过镍柱亲和层析得到纯化的、可溶性的小鼠内皮抑素,经SDS-PAGE分析鉴定。结果高效表达出相对分子量约32KD的重组融合蛋白,灰度扫描软件分析显示,其表达量占菌体总蛋白质的35.9%,经镍柱亲和层析获得了高纯度的小鼠内皮抑素重组融合蛋白,纯度可达95%,得率为0.27g/L。结论用硫氧还蛋白融合表达系统在大肠杆菌中表达的小鼠内皮抑素重组融合蛋白易纯化并具有高活性。

[ Objective ] To express mouse endostatin by using thioredoxin fusion expression system. [Methods ] Endostatin cDNA was amplified by PCR from plasmid pMFGendo, and then cloned into thioredoxin fusion expression vector pThiohisA. The recombinant plasmid was furtherly transformed into E.coli BL21. After induction with IPTG, the product was identified by SDS-PAGE, it was inclusion body. The expression product was purified by affinity chromatography through Ni-column. [Results] The thioredoxin-endostatin fusion protein with a relative molecule mass of 32 KD was highly expressed. Grey-scale analysis showed that the yield of endostatin thioredoxin-endostatin fusion protein was obtained by Ni affinity chromatography fusion protein was 35.9% of the total bacterial protein. The purity of the protein was over 95%. [Conclusion] The mouse endostatin recombinant fusion protein which is expressed in E.coli by using thioredoxin fusion expression system is easy to purify and possess high activity.