Abstract

Chemically modified proteins have diverse applications; however, conventional chemo‐selective methods often yield heterogeneously labeled products. To address this limitation, site‐specific protein labeling holds significant potential, driving extensive research in this area. Nevertheless, site‐specific modification of native proteins remains challenging owing to the complexity of their functional groups. Therefore, a method for site‐selective labeling of intact proteins is aimed to design. In this study, a novel approach to traceless affinity‐directed intact protein labeling is established, which leverages small binding proteins and genetic code expansion technology. By applying this method, a site‐specific antibody labeling with a drug, which leads to the production of highly effective antibody‐drug conjugates specifically targeting breast cancer cell lines is achieved. This approach enables traceless conjugation of intact target proteins, which is a critical advantage in pharmaceutical applications. Furthermore, small helical binding proteins can be easily engineered for various target proteins, thereby expanding their potential applications in diverse fields. This innovative approach represents a significant advancement in site‐specific modification of native proteins, including antibodies. It also bears immense potential for facilitating the development of therapeutic agents for various diseases.