Institute of Biochemistry/Biotechnology, Charles Tanford Protein Centre, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle, Germany.
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120, Halle, Germany.
Chembiochem. 2021 Apr 6;22(7):1201-1204. doi: 10.1002/cbic.202000718. Epub 2021 Jan 15.
Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins' native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.
荧光融合蛋白是研究活细胞中生物过程的有力工具，但由于这些标签体积庞大，可能会影响靶蛋白的折叠、定位甚至生物功能，因此其普遍应用受到限制。设计的生物催化剂 trypsiligase 通过稳定的天然肽键，能够在位于活细胞外膜的完整靶蛋白的 N 端将小尺寸荧光染料定点连接到其上。该方法的功能通过将跨膜蛋白 CD147 以及表皮生长因子受体（均存在于人 HeLa 细胞上）进行共价衍生化的实例进行了测试。通过添加到蛋白质天然 N 端的二肽序列 Arg-His，该方法介导了 trypsiligase 对连接点的特异性识别，该序列指向细胞膜外。如用荧光染料 tetramethyl rhodamine 和亲和标记物 biotin 进行偶联实验所示，标记过程仅需约 5 分钟。
