title: Ultrasensitive Scaffold-Dependent Protease Sensors with Large Dynamic Range.
abstract: The rational construction of synthetic protein switches with predefined input-output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein-protein interactions. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of α-thrombin and prostate specific antigen with a dynamic range covering 5 orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers, and actuators.
authors.affiliation: Institute for Molecular Biosciences, The University of Queensland , QBP Building 80, St Lucia, Queensland 4072, Australia.
authors.forename: Viktor
authors.identifier: 0000-0003-1383-4330
authors.initials: V
authors.lastname: Stein
authors.affiliation: Institute for Molecular Biosciences, The University of Queensland , QBP Building 80, St Lucia, Queensland 4072, Australia.
authors.forename: Masuda
authors.initials: M
authors.lastname: Nabi
authors.affiliation: Institute for Molecular Biosciences, The University of Queensland , QBP Building 80, St Lucia, Queensland 4072, Australia.
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D057089:Pathology, Molecular / Q000379:methods; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D011506:Proteins; D010447:Peptide Hydrolases
keywords: molecular diagnostics; proteases; protein engineering; protein switches; synthetic biology