Title |
Intracellular Detection and Evolution of Site-Specific Proteases Using a Genetic Selection System
|
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Published in |
Applied Biochemistry and Biotechnology, January 2012
|
DOI | 10.1007/s12010-011-9522-6 |
Pubmed ID | |
Authors |
Kathryn D. Verhoeven, Olvia C. Altstadt, Sergey N. Savinov |
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. |
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Student > Master | 5 | 9% |
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Student > Doctoral Student | 3 | 5% |
Other | 7 | 13% |
Unknown | 12 | 21% |
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Nursing and Health Professions | 1 | 2% |
Other | 4 | 7% |
Unknown | 12 | 21% |