Labeling Proteins by Affinity-guided DMAP Chemistry
Labeling Proteins by Affinity-guided DMAP Chemistry
复制标题
通过亲和力引导的 DMAP 化学标记蛋白质
DOI：
10.1007/978-1-4939-2272-7_16
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发表时间：
2015
影响因子：
--
通讯作者：
Itaru Hamachi
中科院分区：
文献类型：
--
作者：
Tomonori Tamura;Itaru Hamachi
催化剂长期以来在有机合成中发挥着重要作用，因此具有作为化学蛋白质修饰工具的潜力。然而，在生物条件下催化剂介导的蛋白质标记的例子很少，因为难以设计在水性环境中具有高靶选择性和反应效率的分子催化剂。为了克服这种情况，我们以前已经开发了一种新的基于催化剂的方法，称为亲和力引导的DMAP（4-二甲基氨基吡啶）（AGD）化学，用于使用酰基转移反应以靶向选择性方式进行位点特异性蛋白质标记。最近，我们发现通过使用“多价”DMAP基团可以大大提高标记速率和效率。在这里，我们描述了多价AGD化学的原理和在试管中化学标记FK 506结合蛋白12（FKBP 12）的方案。在该方法中，FKBP 12标记在30分钟内完成，并且在蛋白质的配体结合口袋附近特异性地发生。
Catalysts have long played an essential role in organic synthesis and thus hold potential as tools for chemical protein modification. However, there are only a few examples of catalyst-mediated protein labeling under biological conditions because of the difficulty of designing molecular catalysts that work in aqueous environments with high target selectivity and reaction efficiency. To overcome this situation, we have previously developed a new catalyst-based method, termed affinity-guided DMAP (4-dimethylaminopyridine) (AGD) chemistry, for site-specific protein labeling in a target-selective manner using an acyl transfer reaction. More recently, we discovered that the labeling rate and efficiency can be greatly enhanced by using “multivalent” DMAP groups. Here, we describe the principle of the multivalent AGD chemistry and the protocol for chemical labeling of FK506-binding protein 12 (FKBP12) in test tubes. In this method, the FKBP12 labeling is completed within 30 min and occurs site specifically at the vicinity of the ligand-binding pocket of the protein.