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在活细胞中使用标记为zinpyr的融合蛋白来检测有机体特异性

Elisa Tomat¹, Elizabeth M Nolan, Jacek Jaworski

¹Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Journal of the American Chemical Society

|November 1, 2008

概括

研究人员开发了一种新方法,使用光探针跟踪活细胞中的含量. 这种蛋白质标记技术成功地定位了线粒体和戈尔吉装置中的传感器,实时显示其对的反应.

科学领域:

细胞生物学细胞生物学
生物化学生物化学
分子成像学分子成像学

背景情况:

准确地定位细胞组件对于理解生物过程至关重要.
是一种必不可少的微量元素,参与了许多细胞功能.
现有的检测细胞内的方法在特异性或实时监测方面可能存在局限性.

研究的目的:

开发和验证一种新的蛋白质标记策略,用于可视化活细胞特定器官中的响应光探针.
为了证明工程探测器在细胞环境中的成功准和响应能力.

主要方法:

传感器 (ZP1) 与基素部分的功能化,以创建人类DNA修复酶基氨酸转移酶 (AGT,也称为SNAP-Tag) 的基质 (ZP1BG).
在体外验证净化AGT和ZP1BG之间的标记反应,并确认由蛋白质结合传感器产生的反应.
在活着的HeLa细胞中测试该系统,这些细胞被设计为表达AGT,并与有机体向蛋白质 (线粒体和戈尔吉器官) 融合.
使用光显微镜来确认传感器的位置.

主要成果:

与蛋白质结合的光检测系统成功地将传感器ZP1BG定位到活的HeLa细胞中的线粒体和Golgi装置.
蛋白质结合传感器的标记反应和反应性在体外和活细胞中得到证实.
工程系统在目标有机体内显示出的反应性.

结论:

一种新的蛋白质标记方法有效地准和可视化活细胞特定器官中的响应的光探针.
这种方法为研究亚细胞区中的动态提供了强大的工具.
开发的系统为活细胞成像应用提供了对有反应的光检测平台.

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