Gerlach Marcus, Stoschek Tina, Leonhardt Heinrich, Hackenberger Christian P R, Schumacher Dominik, Helma Jonas
Department of Biology II, LMU Munich, Planegg/Martinsried, Germany.
Department of Chemical-Biology, Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Methods Mol Biol. 2019;2012:327-355. doi: 10.1007/978-1-4939-9546-2_17.
Tubulin tyrosine ligase (TTL) catalyzes the addition of tyrosine derivatives to the C-terminal carboxylic acid of proteins. The enzyme binds to a 14-amino acid recognition sequence, termed Tub-tag, and allows for the introduction of tyrosine derivatives that carry a unique chemical handle. These handles enable subsequent bioorthogonal reactions with a great variety of probes or effector molecules. Clearly, this two-step chemoenzymatic approach, facilitates the site-specific functionalization of proteins. Furthermore, due to its broad substrate tolerance, tubulin tyrosine ligase also enables an enzymatic one-step modification. For example, a coumarin amino acid was utilized to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics. Here we describe the modification of proteins using TTL in detail via a one-step as well as two-step procedure and highlight its practicability for applications in imaging, diagnostics, and cell biology.
微管蛋白酪氨酸连接酶（TTL）催化将酪氨酸衍生物添加到蛋白质的C末端羧酸上。该酶与一个14个氨基酸的识别序列（称为微管标签）结合，并允许引入带有独特化学基团的酪氨酸衍生物。这些基团能够与多种探针或效应分子进行后续的生物正交反应。显然，这种两步化学酶促方法有助于蛋白质的位点特异性功能化。此外，由于其广泛的底物耐受性，微管蛋白酪氨酸连接酶还能实现酶促一步修饰。例如，一种香豆素氨基酸被用于生成荧光标记的蛋白质，用于成像和诊断等先进应用。在这里，我们详细描述了通过一步法和两步法使用TTL对蛋白质进行修饰，并强调了其在成像、诊断和细胞生物学应用中的实用性。
