ATF-PAI2CD融合蛋白的生物学功能  

Biological Function of Fusion Protein ATF-PAI2CD

作　　者：王霞[1] 李平[1] 张宇清[1] 侯敏[1] 孙兴会[1] 谭理[1] 朱运松[1] 

机构地区：[1]复旦大学上海医学院分子遗传教研室,上海200032

出　　处：《生物化学与生物物理学报》2003年第7期624-628,共5页

基　　金：国家自然科学基金资助项目 (No .3 0 170 3 98)~~

摘　　要：为了解尿激酶型纤溶酶激活物 (urokinase typeplaminogenactivator,uPA)的氨基末端片段 (amino terminalfrag ment,ATF)和纤溶酶激活物抑制剂 2 (plasminogenactivatorinhibitortype 2 ,PAI 2 )突变体PAI 2CD融合蛋白在大肠杆菌的表达情况及进一步研究其生物学活性、将ATF PAI2CD融合基因与大肠杆菌表达载体pLY 4重组得到表达质粒pZWE ATF PAI2CD ,以其转化大肠杆菌JF112 5 ,经温度诱导 ,ATF PAI2CD获得较高水平表达 ,融合蛋白质以包涵体形式存在 ,占菌体总蛋白质的 15 %。包涵体经洗涤、8mol L尿素溶解、稀释复性及离子交换色谱一步分离 ,纯度达 90 % ,分子量与理论值相符。每升发酵液得重组融合蛋白质 5 0mg。经牛奶板法检测具有纤溶抑制活性 ,比活性达 12 0 0 0IU mg ;应用放射竞争法证实融合蛋白质能与肿瘤细胞表面的uPA受体特异性结合。双功能融合蛋白质的纤溶抑制活性与野生型PAI 2 (或PAI 2突变体 ,PAI 2CD)基本一致 ,与肿瘤细胞表面的uPA受体的结合能力同pro uPA。To express the fusion protein ATF PAI2CD (urokinase type plasminogen activator amino terminal fragment plasminogen activator inhibitor type 2 with the region inter C and D helices deleted ) gene in E.coli and determine the biological characterization of fusion protein ATF PAI2CD, the cDNA fragment encoding ATF PAI2CD was cloned into the expression vector pLY 4 and transformed into E.coli JF1125. After temperature induction, the expression amount of ATF PAI2CD account for 15% of total bacterial protein. The result was confirmed by Western blot. ATF PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight 62 kD in SDS PAGE. The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg. The PAI activity was measured by chromogenic assay. The purified fusion protein inhibited urokinase type plasminogen activator as measured by milk agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments. The results indicate that the biological characteristics of ATF PAI2CD were very similar to those of the wide type PAI 2 (or mutants PAI 2, PAI 2CD) and to pro uPA in binding to uPAR bearing cells.

关 键 词：PAI-2 纤溶酶 酶激活 受体 融合蛋白质 表达 肿瘤细胞 比活性 包涵体 突变体 

分 类 号：Q51[生物学—生物化学]