State Key Laboratory of Drug Research, Center for Biotherapeutics Discovery Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No.555 Zuchongzhi Rd, Pudong, Shanghai, 201203, China.
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China.
Nat Commun. 2023 Nov 7;14(1):7169. doi: 10.1038/s41467-023-42977-x.
The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is developed by fusing a cysteine protease domain on the C-terminus of the protein of interest, which could achieve the non-enzymatic C-terminal functionalization by InsP-triggered cysteine protease domain self-cleavage. This method demonstrates a highly efficient way to achieve protein C-terminal functionalization and is compatible with a wide range of amine-containing molecules and proteins. Additionally, a reversible C-terminal de-functionalization is found by incubating the C-terminal modified proteins with cysteine protease domain and InsP, providing a tool for protein functionalization and de-functionalization. Last, various applications of protein C-terminal functionalization are provided in this work, as demonstrated by the site-specific assembly of nanobody drug conjugates, the construction of a bifunctional antibody, the C-terminal fluorescent labeling, and the C-terminal transpeptidation and glycosylation.
精确修饰或功能化蛋白质的 C 末端至关重要，但充满挑战。在此，通过在感兴趣的蛋白质的 C 末端融合半胱氨酸蛋白酶结构域，开发了一种化学方法来修饰 C 末端，该方法可以通过 InsP 触发的半胱氨酸蛋白酶结构域自身切割实现非酶促 C 末端功能化。该方法为实现蛋白质 C 末端功能化提供了一种高效的方法，并且与多种含胺分子和蛋白质兼容。此外，通过将 C 末端修饰的蛋白质与半胱氨酸蛋白酶结构域和 InsP 孵育，发现了可逆的 C 末端去功能化，为蛋白质的功能化和去功能化提供了一种工具。最后，本文提供了蛋白质 C 末端功能化的各种应用，例如通过纳米抗体药物偶联物的定点组装、双功能抗体的构建、C 末端荧光标记以及 C 末端转肽和糖基化来证明。
