Regulation of N-terminal cleavage and splicing in mini-Ter DnaE-3 intein by E23G and R50Q mutations

As a member of the DnaE intein family, Ter DnaE-3 (TE3) is limited by a relatively low splicing rate, significant undesired N-terminal cleavage reactions, and dependence on native exteins.

Building on eight previously identified mutation sites from directed evolution, we employed site-directed mutagenesis to systematically investigate how these residues regulate wild-type mini-TE3 splicing and cleavage activities across various expression systems.

In the pKH kanamycin resistance system, the E23G mutation reduces N-cleavage propensity, which may be attributed to reduced steric hindrance and enhanced structural flexibility. Conversely, in both pKH and pMST systems, the R50Q mutation increases the accumulation of N-terminal cleavage products, suggesting that intein catalysis may be influenced by a distributed regulatory network rather than by isolated residues.

These results suggest that the catalytic behavior of TE3 is governed not only by conserved catalytic cores and adjacent extein sequences but also by distal residues that modulate the intein structure. The discovery that R50Q enhances N-terminal cleavage provides a valuable tool for future protein engineering, particularly for optimizing intein-based protein purification and expressed protein ligation (EPL) technologies.