High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase
High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase
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DOI：
10.1073/pnas.0401722101
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发表时间：
2004-08-10
影响因子：
11.1
通讯作者：
Umezawa, Y
中科院分区：
文献类型：
--
作者：
Kim, SB;Ozawa, T;Umezawa, Y
功能蛋白质的核质运输在响应细胞外信号调节基因表达中起关键作用。我们开发了一种基因编码的生物发光指示剂，用于在体外和体内监测靶蛋白的核运输。该原理是基于通过与DnaE内含肽（DNA聚合酶III的催化亚基）的蛋白质剪接来重建肾形海肾（Rluc）的分裂片段。与Rluc的N-末端一半融合的靶胞质蛋白在哺乳动物细胞中表达。如果蛋白质易位到细胞核中，则Rluc部分与Rluc的C-末端一半相遇，并且全长Rluc通过蛋白质剪接重建。我们证明了基于细胞的定量体外检测配体诱导的雄激素受体易位，这允许高通量筛选外源性和内源性激动剂和拮抗剂。此外，该指示剂能够用电荷耦合器件成像系统对活小鼠脑中的雄激素受体易位进行非侵入性体内成像。这些体外和体内的快速定量分析为筛选药理学或毒理学化合物以及在活体动物中测试它们提供了广泛的应用。
Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.