title: Intracellular detection and evolution of site-specific proteases using a genetic selection system.
abstract: Development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. Difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as therapeutics. Here, we disclose a genetic system, designed to report on site-specific proteolysis through the survival of a bacterial host, and the implementation of this method in the directed evolution of proteases with a non-native substrate preference. The high sensitivity potential of this system was established by monitoring the activity of the Tobacco Etch Virus protease (TEV-Pr) against co-expressed substrates of various recognition level and corroborated by both intracellular and cell-free assays. The genetic selection system was then used in an iterative mode with a library of TEV-Pr mutants to direct the emergence of proteases favoring a nominally poor substrate of the stringently selective protease. The retrieval of mutant enzymes displaying enhanced proteolytic properties against the non-native sequence combined with reduced recognition of the cognate hexapeptide substrate demonstrates the potential of this system for evolving proteases with improved or completely unprecedented properties.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA. kverhoev@umich.edu
authors.forename: Kathryn D
authors.initials: KD
authors.lastname: Verhoeven
authors.forename: Olvia C
authors.initials: OC
authors.lastname: Altstadt
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Savinov
mesh_terms: D001665:Binding Sites; D002470:Cell Survival; D019020:Directed Molecular Evolution / Q000379:methods*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D042541:Intracellular Space / Q000201:enzymology*; D059748:Proteolysis; D013379:Substrate Specificity
chemical_list: D010450:Endopeptidases; C099488:TEV protease