人核迁移蛋白C与血小板生成素受体MPL的结合作用

Binding of Human Nuclear Distribution Protein C to Thrombopoietin Receptor MPL

目的研究人核迁移蛋白C(hNUDC)与血小板生成素受体MPL的结合作用。方法采用PCR技术,分别从人胚胎干细胞和pLexA-MPL-EC质粒中扩增hNUDC和MPL-EC基因,再分别克隆至pET-28b(+)和pMAL-c2表达载体中,转化大肠杆菌BL21(DE3),IPTG诱导表达hNUDC和MPL-EC蛋白。纯化后,通过MBP pull-down试验检测二者的结合作用。结果表达的融合蛋白His-hNUDC和pMAL-MPL-EC表达量分别约占菌体总蛋白的25%和30%,蛋白回收率分别可达90%和80%,纯化后的蛋白纯度分别在95%和90%以上,且均具有良好的反应原性。His-hNUDC蛋白可与MBP-MPL-EC蛋白共洗脱,二者存在结合作用。结论hNUDC蛋白可与MPL蛋白结合,可能为MPL的一种新配体。

Objective To study the binding of human nuclear distribution protein C （hNUDC） to thrombopoietin receptor MPL. Methods hNUDC and MPL-EC genes were amplified by PCR from human embryonic stem cells and plasmid pLexA-MPL-EC, and cloned into expression vectors pET-28b （＋） and pMAL-C2, respectively. The constructed recombinant plasmids were transformed to E. colt BL21 （DE3） for expression under induction of IPTG. The expressed hNUDC and MPL proteins were purified, and their binding was determined by pull-down test. Results The expressed fusion proteins His-hNUDC and pMAL-MPL-EC contained about 25% and 30% of total somatic proteins and reached purities of more than 95% and more than 90% after purification respectively. Both His-hNUDC and pMAL-MPL-EC proteins showed good reactogenieities, and their recovery rates were 90% and 80% respectively. ttis-hNUDC was eluted together with pMAL-MPL-EC, indicating the binding of the 2 kinds of proteins. Conclusion hNUDC protein may be a novel ligand of MPL protein.