Guo Jinbiao, Sun Xiaoyu, Wang Jian, Hou Yimiao, Yang Mingfang, Tan Junjie, Zhang Zhenjie, Chen Yao, Chen Wen
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin, China.
State Key Laboratory of Biopharmaceutical Preparation and Delivery, Chinese Academy of Sciences, Beijing, China.
Nat Commun. 2025 May 3;16(1):4122. doi: 10.1038/s41467-025-59368-z.
Precisely regulating protein conformation (folding) for biomanufacturing and biomedicine is of great significance but remains challenging. In this work, we innovate a covalent organic framework (COF)-directed protein refolding strategy to modulate protein conformation by rationally designed covalent organic frameworks with adapted pore structures and customizable microenvironments. The conformation of denatured protein can be efficiently recovered through a simple one-step approach using covalent organic framework treatment in aqueous or buffer solutions. This strategy demonstrates high generality that can be applied to various proteins (for example, lysozyme, glucose oxidase, trypsin, nattokinase, and papain) and diverse covalent organic frameworks. An in-depth investigation of the refolding mechanism reveals that pore size and microenvironments such as hydrophobicity, π-π conjugation, and hydrogen bonding are critical to regulating protein conformation. Furthermore, we use this covalent organic framework platform to build up solid-phase columns for continuous protein recovery and achieved a ~ 100% refolding yield and excellent recycling performance (30 cycles), enabling an integrated process for the extracting and refolding denatured proteins (such as the harvest of protein in inclusion bodies). This study creates a highly efficient and customizable covalent organic framework platform for precisely regulating proteins refolding and enhancing their performance, opening up a new avenue for advanced protein manufacturing.
精确调控蛋白质构象（折叠）对于生物制造和生物医药具有重要意义，但仍然具有挑战性。在这项工作中，我们创新了一种共价有机框架（COF）导向的蛋白质重折叠策略，通过合理设计具有适配孔结构和可定制微环境的共价有机框架来调节蛋白质构象。变性蛋白质的构象可以通过在水溶液或缓冲溶液中使用共价有机框架处理的简单一步法有效地恢复。该策略具有很高的通用性，可应用于各种蛋白质（例如，溶菌酶、葡萄糖氧化酶、胰蛋白酶、纳豆激酶和木瓜蛋白酶）以及多种共价有机框架。对重折叠机制的深入研究表明，孔径和微环境（如疏水性、π-π共轭和氢键）对于调节蛋白质构象至关重要。此外，我们利用这个共价有机框架平台构建固相柱用于连续蛋白质回收，并实现了约100%的重折叠产率和出色的循环性能（30个循环），从而实现了变性蛋白质提取和重折叠的集成过程（如包涵体中蛋白质的收获）。这项研究创建了一个高效且可定制的共价有机框架平台，用于精确调控蛋白质重折叠并提高其性能，为先进蛋白质制造开辟了一条新途径。
