Yano Yoshiaki, Yano Akiko, Oishi Shinya, Sugimoto Yukihiko, Tsujimoto Gozoh, Fujii Nobutaka, Matsuzaki Katsumi
ACS Chem Biol. 2008 Jun 20;3(6):341-5. doi: 10.1021/cb8000556.
The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of cell-surface receptors using a high affinity coiled-coil formation without metals or enzymes. The peptide probes K3 (KIAALKE)3 and K4 (KIAALKE)4 labeled with a fluorophore specifically stained the surface-exposed tag sequence E3 (EIAALEK)3 attached to the N-terminus of the mouse-derived prostaglandin EP3 receptor in living cells (Kd = 64 and 6 nM for K3 and K4, respectively). The labeling was quick (<1 min), nontoxic, and available even in culture medium without affecting receptor function. As an application of this tractable method, the agonist-induced internalization of the human-derived 2-adrenergic receptor and epidermal growth factor receptor was successfully visualized.
利用可遗传编码的标签和靶向该标签的小型合成探针在活细胞中对蛋白质进行特异性标记，一直是人们梦寐以求的，它可作为广泛使用的较大荧光蛋白的替代方法。我们描述了一种快速方法，该方法使用一个小标签（21个氨基酸），通过形成无金属或酶参与的高亲和力卷曲螺旋来对细胞表面受体进行荧光标记。用荧光团标记的肽探针K3（KIAALKE）3和K4（KIAALKE）4特异性地染色了活细胞中小鼠源前列腺素EP3受体N端连接的表面暴露标签序列E3（EIAALEK）3（K3和K4的解离常数分别为64 nM和6 nM）。标记过程快速（<1分钟）、无毒，甚至在培养基中也可进行，且不影响受体功能。作为这种简便方法的应用，成功实现了人源β2肾上腺素能受体和表皮生长因子受体激动剂诱导的内化的可视化。
