Malawska Katarzyna Joanna, Takano Shinjiro, Oisaki Kounosuke, Yanagisawa Haruaki, Kikkawa Masahide, Tsukuda Tatsuya, Kanai Motomu
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Bioconjug Chem. 2023 Mar 9. doi: 10.1021/acs.bioconjchem.3c00069.
We report the first bioconjugation of Au nanocluster to a monoclonal antibody at scarcely exposed tryptophan (Trp) residues toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the Trp-selective bioconjugation using hydroxylamine (ABNOH) reagents instead of previously developed -oxyl radicals (ABNO). This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure utilizing first Trp-selective bioconjugation for the introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a bicyclononyne (BCN)-presenting redox-sensitive Au nanocluster was essential for a scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au nanocluster conjugates.
我们报道了金纳米团簇首次与单克隆抗体在极少暴露的色氨酸（Trp）残基处进行生物共轭，以开发用于低温电子显微镜（cryo-EM）和断层扫描（cryo-ET）的高分辨率探针。为实现这一目标，我们使用羟胺（ABNOH）试剂改进了色氨酸选择性生物共轭，取代了先前开发的-氧自由基（ABNO）。这种新方案允许将色氨酸选择性生物共轭应用于对酸敏感的蛋白质，如抗体。我们发现，两步法至关重要，第一步是利用色氨酸选择性生物共轭将叠氮基团引入蛋白质，然后通过应变促进的叠氮-炔环加成（SPAAC）连接呈现双环壬炔（BCN）的氧化还原敏感金纳米团簇，以实现可扩展的过程。通过各种分析方法，包括对金纳米团簇共轭物的低温电子显微镜分析，证实了抗体与金纳米团簇的共价标记。
