Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
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DOI：
10.1038/nmeth735
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发表时间：
2005-02-01
期刊：
影响因子：
48
通讯作者：
Ting, AY
中科院分区：
文献类型：
--
作者：
Chen, I;Howarth, M;Ting, AY
我们报告了一种高度特异性、鲁棒性和快速的生物物理探针标记细胞表面蛋白的新方法。该方法使用大肠杆菌生物素连接酶（BirA），其序列特异性地将生物素连接到15个氨基酸受体肽（AP）上。我们报道BirA也接受生物素的酮异构体作为辅助因子，以类似的动力学将该探针连接到AP上，并保持天然反应的高底物特异性。由于天然细胞表面不存在酮，因此可以用酮探针标记ap融合的重组细胞表面蛋白，然后特异性地偶联到肼或羟胺功能化的分子。我们在哺乳动物细胞裂解液和活HeLa细胞表面表达的表皮生长因子受体（EGFR）上展示了这种两阶段蛋白质标记方法。荧光素和二苯甲酮光亲和探针都被纳入，总标记时间短至20分钟。
We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxytamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.