盘状结构域受体2胞外区在酵母系统中的融合表达、纯化和功能鉴定  

Fusion expression and purification of discoidin domain receptor 2 extracellular domain in Pichia pastoris and its functional analysis

作　　者：陈健康[1] 张伟[2] 刘楠楠[1] 刘利兵[1] 田菲[1] 铁茹[1] 

机构地区：[1]第四军医大学教学实验中心,陕西西安710033 [2]第四军医大学微生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2007年第14期1249-1252,共4页Journal of the Fourth Military Medical University

摘　　要：目的:盘状结构域受体2(discoidin domain recep-tor2,DDR2)是一种与肿瘤细胞转移相关的蛋白酪氨酸激酶,研究DDR2在类风湿性关节炎(rheumatoid arthritis,RA)软骨破坏和肿瘤转移中的作用.方法:在毕赤酵母中表达DDR2胞外区片段(不含信号肽和跨膜区,命名为DR),并将所表达的蛋白进行纯化和功能鉴定,以备将来用作DDR2的特异性阻断剂.结果:获得了两株较高表达His-DR融合蛋白的毕赤酵母克隆;其表达的蛋白质中可溶性部分约占全部融合蛋白的18%;经Ni-NTA(nitric-tri-acetic acid)柱纯化后,获得了纯度约90%以上的可溶性His-DR融合蛋白;竞争结合抑制实验显示,His-DR具有阻断和细胞表面天然DDR2受体结合的功能.结论:所表达的融合蛋白His-DR具有抑制天然DDR2功能的作用.AIM： To investigate the role of discoidin domain receptor 2 （DDR2） in the destruction of cartilage in rheumatoid arthritis （RA） and tumor metastasis. METHODS： We expressed extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification （without signal peptide and transmembrane domain, designated DR ） in Pichia pastoris, purified the expressed protein, and characterized its function, for the purpose of future application as a specific DDR2 antagonist. RESULTS： Two clones with relatively high expression of His-DR were obtained. After purification by a NiNTA （nitric-tri-acetic acid ） chromatographic column, soluble fused His-DR with over 90% purity was obtained. Competitive binding inhibition assay demonstrated that the expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on synovial cell surface. CONCLUSION： Extracellular domain of DDR2 fused with a His tag could block the binding of DDR2 and natural DDR2 receptors.

关 键 词：盘状结构域受体2 胞外区 基因表达 阻断剂 

分 类 号：R737.33[医药卫生—肿瘤]