High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology.
High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology.
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DOI：
10.1002/cyto.a.22242
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发表时间：
2013-02
期刊：
影响因子：
3.7
通讯作者：
Sklar, Larry A.
中科院分区：
文献类型：
--
作者：
Wu, Yang;Tapia, Phillip H.;Fisher, Gregory W.;Waggoner, Alan S.;Jarvik, Jonathan;Sklar, Larry A.
在活细胞中同时监测多种蛋白质的运输是非常有趣的，因为发现许多受体蛋白在同一细胞中与其他受体蛋白一起起作用。然而，现有的荧光标记技术限制了功能受体对的机制研究。我们已经扩展了一种结合氟激活蛋白（FAP）技术和高通量流式细胞术的混合系统，使其成为一种新型的生物传感器，它坚固、敏感、用途广泛。这为研究同一细胞中的多种转运蛋白提供了机会。我们选择与FAP AM2.2融合的人β2肾上腺素能受体（β2AR）和与FAP MG13融合的鼠C-C趋化因子受体5型作为模型系统。受体的功能和MG13与氟MG-2p之间的结合已被流式细胞术和共聚焦显微镜测定。氟化物和FAP对的结合具有高度特异性，而这两种FAP标记的融合蛋白的功能与野生型相似。该系统已成功地作为反筛选试验，以消除在NIH分子文库小分子库针对人类β2AR调节因子的筛选中鉴定的假阳性化合物。
Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.
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影响因子：
3.6
作者：
Wu, Yang;Tapia, Phillip H.;Sklar, Larry A.
通讯作者：
Sklar, Larry A.
影响因子：
3.6
作者：
Hoffmann, C;Leitz, MR;Klotz, KN
通讯作者：
Klotz, KN
影响因子：
5.8
作者：
Napier, C;Sale, H;Holbrook, M
通讯作者：
Holbrook, M
DOI：
10.1046/j.1432-1033.2003.03918.x
发表时间：
2004-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Mueller, A;Strange, PG
通讯作者：
Strange, PG
影响因子：
4.4
作者：
Mack, M;Cihak, J;Schlöndorff, D
通讯作者：
Schlöndorff, D