title: Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs.
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.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand Present address: Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
authors.forename: J N
authors.initials: JN
authors.lastname: Copp
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand.
authors.forename: E M
authors.initials: EM
authors.lastname: Williams
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand.
authors.forename: M H
authors.initials: MH
authors.lastname: Rich
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland 1023, New Zealand.
authors.forename: A V
authors.initials: AV
authors.lastname: Patterson
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland 1023, New Zealand.
authors.forename: J B
authors.initials: JB
authors.lastname: Smaill
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand david.ackerley@vuw.ac.nz.
authors.forename: D F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D004249:DNA Damage / Q000187:drug effects; D004269:DNA, Bacterial / Q000737:chemistry / Q000187:drug effects; D019020:Directed Molecular Evolution / Q000379:methods*; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D013014:SOS Response, Genetics
chemical_list: D004269:DNA, Bacterial; D004798:Enzymes; D009153:Mutagens; D011355:Prodrugs
keywords: ADEPT; GDEPT; SOS response; YcnD; YdjA