Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes
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DOI：
10.1038/nbt1355
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发表时间：
2007-12-01
影响因子：
46.9
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
--
作者：
Fernandez-Suarez, Marta;Baruah, Hemanta;Ting, Alice Y.
活细胞成像是研究细胞表面蛋白质动力学的有力手段，但传统的成像探针体积大，或干扰蛋白质功能，或内化后与蛋白质解离。在这里，我们报告了用化学探针对细胞蛋白质进行共价、特异标记的技术。通过合理的设计，我们重定向了微生物硫辛酸连接酶(LPLA)，使其特异性地将烷基叠氮连接到工程LPLA受体肽(LAP)上。然后用不同探针的环辛炔偶合物选择性地衍生烷基叠氮化物。我们用Cy3、Alexa Fluor 568和生物素标记在活的哺乳动物细胞中表达的LAP融合蛋白。我们还将LplA标记与我们之前的生物素连接酶标记相结合，以同时成像在同一细胞中共表达的两个不同受体的动态。我们的方法学应该提供对细胞表面蛋白的生化和成像研究的一般途径，使用通过短肽标签引入的小荧光团。
Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclooctyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.