Copper assisted sequence-specific chemical protein conjugation at a single backbone amide
Mengzhun Guo, Kai Zhao, Liang Guo, Rui Zhou, Qianjun He, Kuan Lu, Tian Li, Dandan Liu, Jinfeng Chen, Jing Tang, Xin Fu, Jinyun Zhou, Bei Zheng, Samuel I. Mann, Yongdeng Zhang, Jing Huang, Bing Yang, Ting Zhou, Yingjie Lei, Bobo Dang
Abstract
Abstract Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose “metal binding targeting” strategy and develop a C opper A ssisted S equence-specific conjugation T ag ( CAST ) method to achieve rapid (second order rate 8.1 M −1 s −1 ), site-specific protein backbone chemical modification with pinpoint accuracy. We demonstrate the versatility of CAST conjugation by preparing various on-demand modified recombinant proteins, including a homogeneous antibody-drug conjugate with high plasma stability and potent efficacy in vitro and in vivo. Thus, CAST provides an efficient and quantitative method to site-specifically attach payloads on large, native proteins.