Kamo Naoki, Hayashi Gosuke, Okamoto Akimitsu
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan.
Methods Mol Biol. 2025;2919:19-45. doi: 10.1007/978-1-0716-4486-7_2.
The chemical synthesis of proteins consists of three steps: synthesis of peptide segments, native chemical ligation, and desulfurization. The native chemical ligation is repeated to grow polypeptides, but at each step, deprotection and purification also had to be repeated. To address these issues, by repeating ruthenium-catalyzed fast deprotection of the cysteine terminus of the peptide segment and slow inactivation of the catalyst by thiophenol step by step, we were able to achieve one-pot, repeated native chemical ligation without purification steps. In this method, the ruthenium catalyst rapidly removes the Alloc group for cysteine protection and is slowly deactivated by 4-mercaptophenylacetic acid, which is added to promote peptide ligation and to remove the allyl group from the ruthenium complex. By using this chemical reaction, we have chemically prepared epigenetically modified proteins such as histone protein H1.2, and the protocol is described here.
肽段合成、天然化学连接和脱硫。重复进行天然化学连接以延长多肽，但在每一步中，脱保护和纯化也都必须重复进行。为了解决这些问题，通过逐步重复钌催化的肽段半胱氨酸末端的快速脱保护以及苯硫酚使催化剂缓慢失活的过程，我们能够实现无需纯化步骤的一锅法重复天然化学连接。在该方法中，钌催化剂能快速去除用于半胱氨酸保护的烯丙氧羰基（Alloc）基团，并被4-巯基苯乙酸缓慢失活，添加4-巯基苯乙酸是为了促进肽连接并从钌配合物中去除烯丙基。通过利用这种化学反应，我们已经化学合成了表观遗传修饰的蛋白质，如组蛋白H1.2，本文将描述该实验方案。
