biostudies-literatureUnknown6(2)Cui TSite-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 JACS Au943-954https://www.ebi.ac.uk/biostudies/studies/S-EPMC12933375biostudies-literature"Lys-Leu" Motif Empowers Expedient &lt;i&gt;Oa&lt;/i&gt;AEP1-Catalyzed Generation of N‑Terminal Cysteine Recombinant Proteins for Bioconjugation and Semisynthesis.PMC12933375Li JLiu NLi YMCui TSun JChu GCfalse"Lys-Leu" Motif Empowers Expedient &lt;i&gt;Oa&lt;/i&gt;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 2026-01-01T00:00:00Z2026 Feb2026-07-16T22:57:44.428Z2026-07-12T03:09:54.396ZS-EPMC129333754175583210.1021/jacsau.5c01395