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.