1.
Protein Eng Des Sel
; 27(10): 399-403, 2014 Oct.
Article
in English
| MEDLINE
| ID: mdl-24996412
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.
Subject(s)
Directed Molecular Evolution/methods , Enzymes/metabolism , High-Throughput Screening Assays/methods , Mutagens/metabolism , Prodrugs/metabolism , Protein Engineering/methods , DNA Damage/drug effects , DNA, Bacterial/chemistry , DNA, Bacterial/drug effects , Enzymes/chemistry , Enzymes/genetics , Enzymes/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Mutagens/chemistry , Mutagens/pharmacology , Prodrugs/chemistry , Prodrugs/pharmacology , SOS Response, Genetics
2.
Br Dent J
; 205(10): 523-4, 2008 Nov 22.
Article
in English
| MEDLINE
| ID: mdl-19023287
Subject(s)
Dental Care for Children/standards , Malpractice , State Dentistry/standards , Child , Humans , United Kingdom
3.
Br Dent J
; 179(10): 372, 1995 Nov 25.
Article
in English
| MEDLINE
| ID: mdl-8519557
4.
Br Dent J
; 178(10): 364, 1995 May 20.
Article
in English
| MEDLINE
| ID: mdl-7779500
5.
Br Dent J
; 161(7): 242, 1986 Oct 11.
Article
in English
| MEDLINE
| ID: mdl-3465341
6.
Br Dent J
; 133(1): 27-9, 1972 Jul 04.
Article
in English
| MEDLINE
| ID: mdl-4507101