The TEV protease S219P (BBa_K316012) is an autocatalysis resistant variant that it’s used to cleave fusion proteins, designed by the iGEM 2010 Imperial College London team. The contributions section on the TEV protease S219P (BBa_K316012) BioBrick registry page provides a detailed description of the work conducted.

We added new documentation to this biobrick, by adding new information about it retrieved from literature and also, we compared the sequences of mutant TEV protease S219P with wild type 1LVM, to determine if TEV S219P exhibits greater efficiency and interaction with its recognition site or synthetic substrate (ENLYFQG), present in the multi peptide sequence that was designed for ALEBBRIGE. Structure-based sequence alignment was performed using Cluster Omega and ESPript3. Afterwards, both enzymes were modeled with AlphaFold for structural analysis, as shown in Figure 1.

Additionally, heatmaps of protein stability for each enzyme were generated using Protein-Sol. Molecular docking was performed with HADDOCK 2.4 and BIOVIA Discovery Studio Visualizer to compare their interactions. A total of 60 images were obtained from the 5 best clusters of the interactions between TEV 1LVM and TEV S219P with their recognition site or synthetic substrate (ENLYFQG). Only those with the highest number of interaction sites were analyzed. Figure 2 presents the analysis of cluster 1 for TEV 1LVM, while Figure 3 shows the analysis of cluster 2 for TEV S219P. All images obtained from the molecular docking can be accessed through the following (link).

The interactions for the 5 clusters of each TEV protease are summarized in Tables 1 and 2. The data indicate that TEV 1LVM exhibited the highest number of interactions, with a total of 105, in contrast to TEV S219P, which displayed a maximum of 64 interactions.

Finally, to corroborate the information obtained from modeling and docking we made our cloning design for TEV S219P in the plasmid pET-24b(+) using Benchling, like it seems in Figure 4.