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

ABSTRACT: 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 OaAEP1-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

SUBMITTER: Cui T 

PROVIDER: S-EPMC12933375 | biostudies-literature | 2026 Feb

REPOSITORIES: biostudies-literature