Protein-protein interaction detection in vitro and in cells by proximity biotinylation
Protein-protein interaction detection in vitro and in cells by proximity biotinylation
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DOI：
10.1021/ja801445p
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发表时间：
2008-07-23
影响因子：
15
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
--
作者：
Fernandez-Suarez, Marta;Chen, T. Scott;Ting, Alice Y.
我们报告了一种在体外和细胞内检测蛋白质相互作用的新方法。其中一个蛋白伴侣与大肠杆菌生物素连接酶（BirA）融合，而另一个蛋白伴侣则与BirA的“受体肽”（AP）底物融合。如果两种蛋白相互作用，BirA将催化AP的位点特异性生物素化，这可以通过链霉亲和素染色检测到。为了最小化非特异性信号，我们设计了AP序列以降低其对BirA的内在亲和力。在体外和细胞中可以检测到雷帕霉素控制的FKBP和FRB蛋白之间的相互作用，信号背景比高达28。我们还将该方法扩展到Cdc25C磷酸酶与14-3-3E磷丝氨酸/苏氨酸结合蛋白之间磷酸化依赖性相互作用的成像。近距离生物素化检测蛋白-蛋白相互作用具有背景低、灵敏度高、AP标签尺寸小、细胞内空间分辨率好等优点。
We report a new method for detection of protein-protein interactions in vitro and in cells. One protein partner is fused to Escherichia coli biotin ligase (BirA), while the other protein partner is fused to BirA's "acceptor peptide" (AP) substrate. If the two proteins interact, BirA will catalyze site-specific biotinylation of AP, which can be detected by streptavidin staining. To minimize nonspecific signals, we engineered the AP sequence to reduce its intrinsic affinity for BirA. The rapamycin-controlled interaction between FKBP and FRB proteins could be detected in vitro and in cells with a signal to background ratio as high as 28. We also extended the method to imaging of the phosphorylation-dependent interaction between Cdc25C phosphatase and 14-3-3E phosphoserine/threonine binding protein. Protein-protein interaction detection by proximity biotinylation has the advantages of low background, high sensitivity, small AP tag size, and good spatial resolution in cells.