title: Intracellular FRET-based Screen for Redesigning the Specificity of Secreted Proteases.
abstract: Proteases are attractive as therapeutics given their ability to catalytically activate or inactivate their targets. However, therapeutic use of proteases is limited by insufficient substrate specificity, since off-target activity can induce undesired side-effects. In addition, few methods exist to enhance the activity and specificity of human proteases, analogous to methods for antibody engineering. Given this need, a general methodology termed protease evolution via cleavage of an intracellular substrate (PrECISE) was developed to enable engineering of human protease activity and specificity toward an arbitrary peptide target. PrECISE relies on coexpression of a protease and a peptide substrate exhibiting Förster resonance energy transfer (FRET) within the endoplasmic reticulum of yeast. Use of the FRET reporter substrate enabled screening large protease libraries using fluorescence activated cell sorting for the activity of interest. To evolve a human protease that selectively cleaves within the central hydrophobic core (KLVF↓F↓AED) of the amyloid beta (Aβ) peptide, PrECISE was applied to human kallikrein 7, a protease with Aβ cleavage activity but broad selectivity, with a strong preference for tyrosine (Y) at P1. This method yielded a protease variant which displayed up to 30-fold improvements in Aβ selectivity mediated by a reduction in activity toward substrates containing tyrosine. Additionally, the increased selectivity of the variant led to reduced toxicity toward PC12 neuronal-like cells and 16-1000-fold improved resistance to wild-type inhibitors. PrECISE thus provides a powerful high-throughput capability to redesign human proteases for therapeutic use.
authors.affiliation: Department of Chemical Engineering, University of California , Santa Barbara, California 93106, United States.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Guerrero
authors.affiliation: Department of Chemical Engineering, University of California , Santa Barbara, California 93106, United States.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: O'Malley
authors.affiliation: Department of Chemical Engineering, University of California , Santa Barbara, California 93106, United States.
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Daugherty
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D010447:Peptide Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010447:Peptide Hydrolases