Intracellular Detection and Evolution of Site-Specific Proteases Using a Genetic Selection System
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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.
KeywordsProteases 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).
- 2.Christie, R. B. (1980). The medical uses of proteolytic enzymes. In A. Wiseman (Ed.), Topics in enzyme and fermentation biotechnology (pp. 25–113). Chichester: Ellis Horwood.Google Scholar
- 7.Kurschus, F. C., & Jenne, D. E. (2010). Delivery and therapeutic potential of human granzyme B. Immunology Reviews, 235, 159–171.Google Scholar
- 8.Stenman, S. M., Venalainen, J. I., Lindfors, K., Auriola, S., Mauriala, T., Kaukovirta-Norja, A., Jantunen, A., Laurila, K., Qiao, S. W., Sollid, L. M., Mannisto, P. T., Kaukinen, K., & Maki, M. (2009). Enzymatic detoxification of gluten by germinating wheat proteases: Implications for new treatment of celiac disease. Annals of Medicine, 41, 390–400.CrossRefGoogle Scholar
- 19.Miller, J. (1972). Experiments in molecular genetics. Cold Spring Harbor Laboratory: Cold Spring Harbor.Google Scholar
- 22.Cadwell, R. C., & Joyce, G. F. (1994). Mutagenic PCR. PCR Methods Applied, 3, S136–S140.Google Scholar
- 29.Lallo, G. D., Castagnoli, L., Ghelardini, P., & Paolozzi, L. (2001). A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli. Microbiology, 147, 1651–1656.Google Scholar
- 34.Khlebnikov, A., Datsenko, K. A., Skaug, T., Wanner, B. L., & Keasling, J. D. (2001). Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology, 147, 3241–3247.Google Scholar
- 37.Mondigler, M., & Ehrmann, M. (1996). Site-specific proteolysis of the Escherichia coli SecA protein in vivo. Journal of Bacteriology, 178, 2986–2988.Google Scholar
- 43.Verhoeven, K.D. (2011) Genetic system for detecting, monitoring, and evolving site-specific proteases: Development of therapeutic proteases targeting amyloid-beta peptide, Ph.D. Thesis, Purdue University, West Lafayette.Google Scholar
- 45.Puhl, A. C., Giacomini, C., Irazoqui, G., Batista-Viera, F., Villarino, A., & Terenzi, H. (2009). Covalent immobilization of tobacco-etch-virus NIa protease: A useful tool for cleavage of the histidine tag of recombinant proteins. Biotechnology and Applied Biochemistry, 53, 165–174.CrossRefGoogle Scholar