Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry
Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry
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DOI：
10.1038/nprot.2011.425
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发表时间：
2012-01-01
期刊：
影响因子：
14.8
通讯作者：
Tate, Edward W.
中科院分区：
文献类型：
--
作者：
Heal, William P.;Wright, Megan H.;Tate, Edward W.
描述了一种用于蛋白质的选择性和位点特异性酶标记的方案。该方法利用了被称为肉豆蔻酰化的蛋白质共翻译/翻译后修饰，肉豆蔻酸（一种14碳饱和脂肪酸）转移到N-末端甘氨酸，由酶肉豆蔻酰-CoA催化：蛋白质N-肉豆蔻酰转移酶（NMT）。不具有内源性NMT的大肠杆菌用于共表达转移酶和待标记的靶蛋白，其参与带有“可点击”标签的合成衍生的带标签的肉豆蔻酸类似物的体内N-末端连接。该标签是可以通过“点击”化学进行生物正交连接的官能团，例如叠氮化物，并且可以用作进一步体外位点特异性标记的手柄。在这里，我们提供了在体内N-末端标记的重组蛋白，和多功能试剂的合成和应用，使蛋白质标记通过点击化学亲和纯化和荧光检测的协议。除了一般的N-末端蛋白质标记之外，该方案将特别用于提供感兴趣的蛋白质的天然豆蔻酰化的证据、NMT的活性/选择性的证据和NMT的跨物种反应性，而不诉诸于使用放射性同位素。
A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA: protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.