光向细胞蛋白通过酶介导的亚酸结合和菌株促进的循环添加
Jennifer Z Yao1, Chayasith Uttamapinant, Andrei Poloukhtine
1Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue. Cambridge, Massachusetts 02139, USA.
Journal of the American Chemical Society
|January 14, 2012
概括
这项研究引入了一种两步方法,用于使用工程酶用各种光体标记细胞蛋白质. 与传统的光蛋白相比,这种酶标记可以实现更明亮,更稳定的成像.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 化学生物学 化学生物学
背景情况:
- 小分子光体在细胞成像中比光蛋白具有优势,包括更小的尺寸,增强的亮度和优越的光稳定性.
- 之前的工作建立了一种蓝色光向蛋白质的方法,使用酸酶 (LplA) 突变体和标记 (LAP).
研究的目的:
- 将基于LplA的酶标签扩展到绿色和红色光体.
- 为各种细胞蛋白质成像开发一个优化的两步标签策略.
主要方法:
- 一种两步的方法,涉及亚基对LAP标签的特定位点的酶结合,然后是无铜的点击化学与环-相结合物.
- 评估不同的循环氨酸衍生物,包括aza-dibenzocyclooctyne (ADIBO) 和单化循环氨酸 (MOFO),以实现高效的相结合.
- 在各种哺乳动物细胞系中应用优化标记方案,以标记LAP融合蛋白.
主要成果:
- LplA的W37I突变有效地将10-azidodecanoic酸与细胞中的LAP标签结合起来.
- ADIBO-光剂提供了高特异性,而MOFO-光剂对于像ATTO 647N.这样的疏水性染料优越.
- 使用广泛的光体,成功成像LAP融合蛋白质,包括光素,罗达胺,Alexa Fluor 568,ATTO 647N和ATTO 655.
结论:
- 一个优化的两步酶和化学标签策略使得哺乳动物细胞中的多功能和高效的特定位点蛋白质标签成为可能.
- 这种方法允许使用广泛的小分子光体,显著提高了细胞成像能力.
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