Yajie Liu^#, Xilin Bai, Chengliang Lyu, Jing Fang, Fan Zhang, Wen-Hao Wu, Wei Wei*, and Wen-Bin Zhang* Mechano-bioconjugation Strategy Empowering Fusion Protein Therapeutics with Aggregation Resistance, Prolonged Circulation, and Enhanced Antitumor Efficacy. J. Am. Chem. Soc. 2022, 144, 18387-18396. https://doi.org/10.1021/jacs.2c06532

Bioconjugation is a powerful protein modification strategy to improve protein properties. Herein, we report mechano-bioconjugation as a novel approach to empower fusion protein therapeutics and demonstrate its utility by a protein heterocatenane (cat-IFN-ABD) containing interferon-α2b (IFN) mechanically interlocked with a consensus albumin-binding domain (ABD). The conjugate was selectively synthesized in cellulo following a cascade of post-translational events using a pair of heterodimerizing p53dim variants and two orthogonal split-intein reactions. The catenane topology was proven by combined techniques of LC−MS, SDS-PAGE, SEC, and controlled proteolytic digestion. Not only did catIFN-ABD retain activities comparable to those of the wild-type IFN and ABD, the conjugate also exhibited enhanced aggregation resistance and prolonged circulation time over the simple linear and cyclic fusions. Consequently, cat-IFN-ABD potently inhibited tumor growth in the mouse xenograft model. Therefore, mechano-bioconjugation by catenation accomplishes function integration with additional benefits, providing an alternative pathway for developing advanced protein therapeutics.