融合蛋白ATF-PAI2CD的生物学功能。

Xia Wang, Ping Li, Yu-Qing Zhang, Min Hou, Xing-Hui Sun, Li Tan, Yun-Song Zhu
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摘要

为了在大肠杆菌中表达融合蛋白ATF-PAI2CD(尿激酶型纤溶酶原激活物氨基末端片段-纤溶酶原激活物抑制剂2型,缺失C和D螺旋区)基因,并确定融合蛋白ATF-PAI2CD的生物学特性,将编码ATF-PAI2CD的cDNA片段克隆到表达载体pLY-4中,转化大肠杆菌JF1125。经温度诱导后,ATF-PAI2CD的表达量占细菌总蛋白的15%。Western blot证实了这一结果。通过包裹体洗涤和增溶、复性和离子交换层析分离纯化ATF-PAI2CD蛋白。最终产物在SDS-PAGE上显示一个分子量为62 kD的单条带。纯度在90%以上,蛋白质收率为50%,比活性为12 000 IU/mg。用显色法测定PAI活性。经乳琼脂糖平板法检测,纯化的融合蛋白对尿激酶型纤溶酶原激活物有抑制作用,并通过uPA受体(uPAR)与人肺癌细胞结合,经无线电竞争实验证实。结果表明,ATF-PAI2CD的生物学特性与宽型PAI-2(或突变体PAI-2、PAI-2CD)和pro-uPA结合携带upa的细胞非常相似。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
[Biological function of fusion protein ATF-PAI2CD].

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.

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