Applied Biochemistry and Biotechnology

, Volume 166, Issue 5, pp 1340–1354 | Cite as

Intracellular Detection and Evolution of Site-Specific Proteases Using a Genetic Selection System

  • Kathryn D. VerhoevenEmail author
  • Olvia C. Altstadt
  • Sergey N. Savinov


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.


Proteases Genetic reporter Protein engineering Genetic selection Directed evolution 



The authors would like to thank B. L. Wanner (Purdue University, West Lafayette, Indiana) for donation of strains and plasmids. This study was sponsored by the National Institutes of Health (grant number: 1R21AG031437-01).

Supplementary material

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Kathryn D. Verhoeven
    • 1
    Email author
  • Olvia C. Altstadt
    • 1
  • Sergey N. Savinov
    • 1
  1. 1.Department of ChemistryPurdue UniversityWest LafayetteUSA

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