Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing
Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing
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DOI：
10.1021/ac0013296
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发表时间：
2001-06-01
影响因子：
7.4
通讯作者：
Umezawa, Y
中科院分区：
文献类型：
--
作者：
Ozawa, T;Kaihara, A;Umezawa, Y
我们描述了一种新的方法，用于检测完整的哺乳动物细胞中的蛋白质-蛋白质相互作用，该方法是基于蛋白质剪接诱导的萤火虫荧光素酶的合理设计的片段的互补。蛋白质剪接是一种翻译后蛋白质修饰，通过这种修饰，内含肽（内部蛋白质）从前体融合蛋白中切除，将侧翼的外显肽（外部蛋白质）连接成连续的多肽。作为内含肽，使用来自集胞藻属（Synechocystis sp）PCC 6803的天然分裂的DnaE：将各自分别与分裂荧光素酶的N-和C-末端片段融合的N-和C-末端DnaE连接至感兴趣的蛋白质。在这种方法中，蛋白质-蛋白质相互作用触发DnaE内含肽的折叠，其中发生蛋白质剪接，从而连接的荧光素酶的外显肽恢复其酶活性。为了测试这种分裂荧光素酶互补的适用性，我们使用已知的结合伴侣，磷酸化胰岛素受体底物1（IRS-1）和其靶向PI 3-激酶的N-末端SH 2结构域之间的胰岛素诱导的相互作用。利用胰岛素诱导的荧光素酶活性，以胰岛素剂量依赖的方式监测IRS-1与SH 2结构域的相互作用，并通过荧光强度来评估其量。这为研究任何蛋白质的磷酸化或膜蛋白的相互作用提供了一种方便的方法，而这类分子一直难以用现有的生物化学或遗传学方法进行研究。该系统还可用于高通量药物筛选和胰岛素信号转导中IRS-1酪氨酸磷酸化特异性途径的定量分析。
We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly Luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp, PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of Ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin sen-ed to monitor the interaction bem een IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity, This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.