Precise modulation of protein refolding by rationally designed covalent organic frameworks.

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.