Site-specific protein labeling using PRIME and chelation-assisted click chemistry.
Site-specific protein labeling using PRIME and chelation-assisted click chemistry.
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DOI：
10.1038/nprot.2013.096
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发表时间：
2013-08
期刊：
影响因子：
14.8
通讯作者：
Ting AY
中科院分区：
文献类型：
--
作者：
Uttamapinant C;Sanchez MI;Liu DS;Yao JZ;Ting AY
该协议描述了一种用化学探针对细胞表面或纯化蛋白质进行位点特异性标记的有效方法，分两步：酶介导的探针掺入（PRIME），然后是螯合辅助铜催化叠氮炔环加成（CuAAC）。在 PRIME 步骤中，大肠杆菌硫辛酸连接酶将吡啶甲基叠氮衍生物位点特异性连接到已通过基因方法融合到目标蛋白质上的 13 个氨基酸识别序列上。在第二步中，带有吡啶甲基叠氮化物的蛋白质通过螯合辅助的 CuAAC 与炔烃-探针缀合物进行化学选择性衍生。我们在此描述了合成吡啶甲基叠氮化物、进行 PRIME 标记以及在活细胞、固定细胞和纯化蛋白质上实现吡啶甲基叠氮化物的 CuAAC 衍生化的优化方案。试剂制备，包括吡啶甲基叠氮化物探针的合成和硫辛酸连接酶的表达，需要12天，而对细胞或纯化蛋白进行位点特异性吡啶甲基叠氮化物连接和CuAAC的过程需要40分钟至3小时。
This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: PRobe Incorporation Mediated by Enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase site-specifically attaches a picolyl azide derivative to a 13-amino acid recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing picolyl azide are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize picolyl azide, perform PRIME labeling, and achieve CuAAC derivatization of picolyl azide on live cells, fixed cells, and purified proteins. Reagent preparations, including synthesis of picolyl azide probes and expression of lipoic acid ligase, take 12 d, while the procedure to perform site-specific picolyl azide ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.