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- Title
Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs.
- Authors
Copp, J.N.; Williams, E.M.; Rich, M.H.; Patterson, A.V.; Smaill, J.B.; Ackerley, D.F.
- Abstract
Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90 000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background.
- Subjects
HIGH throughput screening (Drug development); GENETIC toxicology; NITROREDUCTASES; PRODRUGS; ANTINEOPLASTIC agents; DNA damage
- Publication
PEDS: Protein Engineering, Design & Selection, 2014, Vol 27, Issue 10, p399
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzu025