GFP reporter screens for the engineering of amino acid degrading enzymes from libraries expressed in bacteria.
Methods Mol Biol
; 978: 31-44, 2013.
Article
em En
| MEDLINE
| ID: mdl-23423887
ABSTRACT
There is significant interest in engineering human amino acid degrading enzymes as non-immunogenic chemotherapeutic agents. We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporter to the catalytic activity of the desired amino acid degrading enzyme in an appropriate E. coli genetic background. The method described here allows facile screening of much larger libraries (10(6)-10(7)) than was previously possible. We demonstrate the application of this technique in the screening of libraries of bacterial and human asparaginases and also for the catalytic optimization of an engineered human methionine gamma lyase.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia de Proteínas
/
Proteínas de Fluorescência Verde
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos