Trypsiligase-Catalyzed Labeling of Proteins on Living Cells.
Trypsiligase-Catalyzed Labeling of Proteins on Living Cells.
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DOI：
10.1002/cbic.202000718
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发表时间：
2021-04-06
期刊：
影响因子：
--
通讯作者：
Bordusa F
中科院分区：
文献类型：
--
作者：
Liebscher S;Mathea S;Aumüller T;Pech A;Bordusa F
荧光融合蛋白是研究活细胞生物过程的有力工具，但由于这些标签的体积庞大，可能会影响目标蛋白的折叠、定位甚至生物功能，因此普遍应用受到限制。设计的生物催化剂胰蛋白酶连接酶能够通过稳定的天然肽键将小尺寸荧光染料定点连接到位于活细胞外膜的整体目标蛋白的 N 末端。通过使用人类 HeLa 细胞上存在的跨膜蛋白 CD147 和 EGF 受体的共价衍生化示例来测试该方法的功能。胰酶连接酶对连接位点的特异性识别是由添加到蛋白质天然 N 末端（指向细胞膜外）的二肽序列 Arg-His 介导的。标记过程仅需约 5 分钟，荧光染料四甲基罗丹明和亲和标记生物素的偶联也证明了这一点。 荧光融合蛋白：在此，我们描述了一种通过胰蛋白酶连接细胞表面结合蛋白进行位点特异性生物共轭的生物催化方法。该反应快速、不可逆、无副反应且对活细胞完全无毒。重要的是，胰蛋白酶连接酶识别靶膜蛋白只需要在其 N 末端引入简单的二肽序列。
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. Fluorescent fusion proteins: Herein, we describe a biocatalytic approach for the site‐specific bioconjugation of cell‐surface bound proteins by trypsiligase. The reactions are fast, irreversible, without side reactions and completely nontoxic to living cells. Importantly, recognition of the target membrane protein by trypsiligase only requires the introduction of a simple dipeptide sequence at its N terminus.
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