公开(公告)号：US07700375B2

公开(公告)日：2010-04-20

申请号：US11153398

申请日：2005-06-16

申请人： Jeffrey W. Keillor ,  Stephen W. Michnick ,  Stéphane Girouard

发明人： Jeffrey W. Keillor ,  Stephen W. Michnick ,  Stéphane Girouard

IPC分类号： G01N33/533 ,  G01N33/53

CPC分类号： G01N33/582

摘要： New methods are provided for the post-genomic era that will permit the analysis of the dynamic expression and localization of gene products in living cells. Herein we propose the development of such a method from a bioorganic approach involving organic synthesis and protein engineering. Specifically, novel compounds bearing two maleimide groups attached directly to fluorescent cores will be prepared, whose latent fluorescence is quenched until their maleimide groups undergo a specific thiol addition reaction. Complementary a-helical proteins are designed bearing two cysteine residues appropriately positioned to react with our novel fluorogens. Genetically fusing our helical probe peptides to test proteins of interest, we can selectively label the target sequence in living cells with our small synthetic fluorogenic molecules. The scope of this technique is described in the context of studying protein localization and protein-protein interactions in living cells.

摘要翻译： 为基因组后时代提供了新的方法，可以分析活细胞中基因产物的动态表达和定位。 在这里，我们提出从涉及有机合成和蛋白质工程的生物有机方法开发这种方法。 具体地说，将制备直接连接到荧光核心上的具有两个马来酰亚胺基团的新型化合物，其潜伏荧光骤冷直到其马来酰亚胺基团进行特定的硫醇加成反应。 设计了互补的α-螺旋蛋白，其具有适当定位以与我们的新型氟原子反应的两个半胱氨酸残基。 基因上融合我们的螺旋探针肽来测试感兴趣的蛋白质，我们可以用我们的小合成荧光分子选择性地标记活细胞中的靶序列。 在研究活细胞中蛋白质定位和蛋白质 - 蛋白质相互作用的背景下描述了该技术的范围。