A Unique THN Motif Is Critical for Enabling Efficient C‐Terminal Traceless Cleavage

Abstract Traceless protein cleavage is a significant challenge in intein application, as most common inteins studied today are not both active and promiscuous. In this study, the intein gp41‐1 is engineered, which demonstrates the most efficient traceless cleavage reported to date and shows high compatibility to 1st amino acid. The evidence provided for the first time is that the unique THN motif, which is prevalent in class 3 inteins, is essential for achieving high‐efficiency traceless C‐terminal cleavage. The hydrogen bond between the hydroxyl group of Thr ₁₂₃ and the main chain of His ₁₂₄ is suggested to be indispensable for stabilizing the THN motif to separate Asp ₁₀₇ (the limiting factor for C‐cleavage) from Asn ₁₂₅ and the C‐extein residues from the active sites, which jointly lead to the highest traceless C‐cleavage activity. Both cleavage data and molecular dynamics (MD) simulations results demonstrate that mutating Thr ₁₂₃ greatly disturbed the THN motif, leading to inactivity. These findings reveal a pivotal motif for intein traceless cleavage efficiency, providing valuable insights for designing inteins with enhanced traceless C‐terminal cleavage capabilities in future applications.