3.5 Protease evolution via cleavage of an intracellular substrate (PrECISE).
In parallel to the YESS system, Guerrero and coworkers developed an ER sequestration approach that relies on intracellular FRET release, which they named protease evolution via cleavage of an intracellular substrate (PrECISE) (Guerrero et al., 2016). In PrECISE, FRET is released upon protease cleavage of a linker sequence that keeps a quenched two-protein FRET pair (Figrue 3D). To showcase PrECISE, they evolved the cleavage specificity of human kallikrein 7 (hK7) towards the amyloid-β8 sequence KLVFFAED (Aβ8). Cleavage of Aβ after phenylalanine within Aβ8 disrupts its aggregation. However, wild type hK7 shows little activity towards Aβ8. Using error-prone PCR, hK7 was engineered to yield variant hK7-2.7, harboring three mutations compared to WT hK7. Variant hK7’s switched specificity was primarily driven by a decreased preference towards tyrosine at the P1 position compared to WT hK7 and not an increased preference for phenylalanine at P1. Curiously, hK7-2.7’s catalytic efficiency Aβ8 was only ~80% of that of WT on that same sequence. The selection of a variant with lower catalytic efficiency on the positive selection substrate is unexpected. Without a dedicated counterselection substrate, the evolution campaign selected against promiscuous variants that impacted yeast cell growth. As evidence, the authors find that yeast cells harboring evolved variants outgrew those with the wild-type enzyme. The growth selection aspect of cell-based screens may be leveraged to narrow the substrate specificity of toxic enzymes.