二聚化依赖型绿色和黄色荧光蛋白。
Dimerization-dependent green and yellow fluorescent proteins.
作者信息
Alford Spencer C, Ding Yidan, Simmen Thomas, Campbell Robert E
出版信息
ACS Synth Biol. 2012 Dec 21;1(12):569-75. doi: 10.1021/sb300050j. Epub 2012 Aug 14.
Abstract
Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pK a for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.
摘要
二聚化依赖性荧光蛋白（ddFP）是最近引入的一类基因编码报告分子，可用于检测活细胞中的蛋白质相互作用。这类工具的前身是一种红色荧光ddFP（ddRFP），它源自盘基网柄菌红色荧光蛋白的同型二聚体变体。在此，我们描述了一个扩展的ddFP调色板的工程设计和应用，其中包括绿色（ddGFP）和黄色（ddYFP）变体。这些优化后的变体相对于ddRFP具有几个优点，包括ddGFP在体外具有更高的对比度和亮度，以及ddYFP具有更高的亮度和更低的pKa。我们证明这两种变体都可作为活细胞中蛋白酶活性的生物传感器。使用ddGFP工具，我们生成了一种用于内质网（ER）和线粒体的线粒体相关膜（MAM）界面成像的高效内膜接近度指示剂。
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