Sarpong Kwabena, Bose Ron
Division of Oncology, Department of Medicine, Washington University School of Medicine, Campus Box 8076, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Division of Oncology, Department of Medicine, Washington University School of Medicine, Campus Box 8076, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Anal Biochem. 2017 Mar 15;521:55-58. doi: 10.1016/j.ab.2017.01.008. Epub 2017 Jan 11.
A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.
将荧光团或其他连接基团连接到蛋白质上的一个主要挑战是要有一种位点特异性标记策略，该策略能实现化学计量修饰而不损害蛋白质的完整性。我们开发了一种简单的方法，将烟草蚀纹病毒（TEV）蛋白酶切割、分选酶修饰和亲和纯化相结合，用于蛋白质的N端标记。为了获得化学计量标记的蛋白质，我们在含荧光团的肽中加入了一个短亲和标签，用于标记后修饰蛋白质的纯化。这种策略可以很容易地应用于任何具有TEV位点的重组蛋白，我们在表皮生长因子受体（EGFR）和膜支架蛋白（MSP）构建体上证明了这一点。
