YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants

Here we describe YESS 2.0, a highly versatile version of the yeast endoplasmic sequestration screening (YESS) system suitable for engineering and characterizing protein/peptide modifying enzymes such as proteases with desired new activities. By incorporating features that modulate gene transcription well substrate enzyme spatial sequestration, 2.0 achieves significantly higher operational dynamic range compared original YESS. To showcase advantages improved an already efficient TEV protease variant (TEV-EAV) to obtain (eTEV) 2.25-fold catalytic efficiency, derived almost entirely from increase in turnover rate (kcat). In our analysis, eTEV specifically digests fusion protein 2 h at low 1:200 ratio. Structural modeling indicates efficiency is likely due enhanced interaction between Cys151 P1 residue (Gln). Furthermore, showed ENLYFQS peptide buried larger extent active site WT TEV. The functionally fastest reported date could potentially improve any application.