Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands.
Chembiochem. 2022 Jun 3;23(11):e202200144. doi: 10.1002/cbic.202200144. Epub 2022 Apr 19.
Methods for facile site-selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site-specific covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein. Although this Flavin-tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin-tag method that addresses previous limitations. We demonstrate that co-expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin-labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable.
方法便于选择的蛋白质的修饰是有很高的需求。我们最近表明，黄素转移酶可以用于定点共价连接一个发色团和荧光黄素（FMN）的任何目标蛋白。虽然这种黄素标签法导致蛋白质在体外的有效标记，标记大肠杆菌细胞中导致部分黄素掺入。它也局限于安装标签的类型，只有一个类型的黄素，FMN，被掺入。在这里，我们报告了黄素标签法的扩展，解决了以前的限制。我们证明，共表达的 FAD 合成酶提高了黄素掺入效率，允许在大肠杆菌细胞中完全黄素标记的目标蛋白。此外，我们已经发现，各种黄素衍生物，甚至烟酰胺可以共价连接到一个目标蛋白，使这种方法更加通用和有价值。
