Engineering the Tobacco Etch Virus Protease toward a Platform for Traceless Cleavage Using Distal Site Prediction and Smart Library Design.

Protein tags are vital in biochemical engineering but must be removed from target molecules to prevent compromising effects. Most industrial applications use Tobacco Etch Virus protease (TEVp) for this purpose. However, selectivity at the P1' position of its recognition site requires N-terminal addition of glycine or serine to noncanonical targets. This residue remains after cleavage, preventing the retrieval of customized peptides with native N-termini. Here, we engineered a TEVp variant (TEVp-C1) with unlocked promiscuity informed by graph network analysis to identify distal positions of influence and smart library design. Compared to state-of-the-art, TEVp-C1 exhibits significantly improved cleavage for 15 of the 20 natural amino acids at P1' against Switchtag-Teriparatide substrates. Moreover, TEVp-C1 displayed enhanced activity against fluorogenic peptide substrates for five of the most disfavored residues at P1'. Mechanistic analysis revealed that the introduced mutations facilitate the proton transfer step. Altogether, the results highlight the potential of TEVp-C1 as a protease platform for traceless cleavage and demonstrate the feasibility of using tools for the prediction of allosteric interactions to engineer substrate specificities of enzymes via mutations at distal amino acid positions.