National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, College of Future Technology, Peking University, Yiheyuan Road No.5, Beijing, 100871, China.
PKU-Nanjing Institute of Translational Medicine, Nanjing, 211800, Jiangsu, China.
Angew Chem Int Ed Engl. 2022 Sep 5;61(36):e202207905. doi: 10.1002/anie.202207905. Epub 2022 Jul 29.
Self-labeling protein tags can introduce advanced molecular motifs to specific cellular proteins. Here we introduce the third-generation covalent TMP-tag (TMP-tag3) and showcase its comparison with HaloTag and SNAP-tag. TMP-tag3 is based on a proximity-induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)-acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP-tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18-kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal N-C termini of eDHFR also enable facile insertion into various protein loops. TMP-tag3, HaloTag, and SNAP-tag are orthogonal to each other, collectively forming a toolbox for multiplexed live-cell imaging of cellular proteins under fluorescence nanoscopy.
自标记蛋白标签可以将先进的分子基序引入特定的细胞蛋白中。在这里，我们引入了第三代共价 TMP 标签（TMP-tag3），并展示了它与 HaloTag 和 SNAP-tag 的比较。TMP-tag3 基于工程化的大肠杆菌二氢叶酸还原酶（eDHFR）中的一个 Cys 与优化的三甲基嘧啶（TMP）-丙烯酰胺缀合物之间的近程诱导共价迈克尔加成，缀合物的连接子最短。与之前的版本相比，TMP-tag3 与荧光螺环罗丹明缀合时具有增强的通透性。作为一种小蛋白，18kD 的 eDHFR 有利于标记选择的线粒体蛋白，这些蛋白与更大的 HaloTag 融合体不太兼容。eDHFR 的近端 N-C 末端也便于插入各种蛋白质环中。TMP-tag3、HaloTag 和 SNAP-tag 彼此正交，共同构成了荧光纳米显微镜下细胞蛋白的多路复用活细胞成像工具盒。
