“Lys–Leu” Motif Empowers Expedient <i>Oa</i> AEP1-Catalyzed Generation of N-Terminal Cysteine Recombinant Proteins for Bioconjugation and Semisynthesis

Site-specific protein modification enables precise functionalization for applications ranging from live-cell imaging to therapeutic development. Although N-terminal cysteine (N-Cys)-based modification exhibits excellent specificity, naturally occurring N-Cys is rare, and existing methods are usually limited by factors such as narrow substrate scopes, incompatibility with inclusion bodies, or residual exogenous sequences. Here, we report a highly efficient and versatile method for generating free N-Cys proteins through "Lys-Leu" (K-L) motif-assisted <i>Oa</i>AEP1-catalyzed cleavage. This approach enhances cleavage efficiency across all 20 natural amino acids at the P2' position, accommodates diverse substrates (including challenging inclusion body proteins), and enables streamlined one-pot in situ generation and functionalization, as exemplified by fluorescein/biotin labeling and dual modifications. Furthermore, we successfully applied this strategy to the traceless semisynthesis of histone H3Q5ser (serotonylated at Gln5). Our work establishes a robust platform for site-specific protein engineering and expands the functional versatility of <i>Oa</i>AEP1 in synthetic biology and chemical biology applications.