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Biotechnol Prog ; 26(6): 1765-71, 2010.
Article in English | MEDLINE | ID: mdl-20574990

ABSTRACT

A genetic Fluorescence Resonance Energy Transfer (FRET) detector undergoes a post-translational modification (PTM)-induced conformational change that results in increased FRET. To test if the PTM-dependent FRET change can be quantified by flow cytometry, we purified and immobilized a genetic detector on microbeads and used flow cytometry to measure its FRET efficiency before and after Erk-2-mediated phosphorylation. The fluorescence ratio R between the acceptor and donor fluorescence, which was obtained by fitting a straight line through the data points in linear space, increases following phosphorylation, thus demonstrating that flow cytometry is capable of detecting a PTM-dependent FRET response. Furthermore, when Erk-2 and a genetic detector are coexpressed in bacteria, the measured R value changes with the substrate sequence with near single residue resolution. Similarly, the cells coexpressing the glycosylating enzyme O-GlcNAc transferase (OGT) and a genetic detector specific for OGT exhibit a PTM-induced change in FRET efficiency. Therefore, the combination of flow cytometry and a genetic detector may be useful to characterize the substrate specificity of a PTM enzyme and identify the sequences that are preferentially targeted for PTM in vivo.


Subject(s)
Flow Cytometry/methods , Fluorescence Resonance Energy Transfer , Mitogen-Activated Protein Kinase 1/metabolism , Amino Acid Sequence , Genetic Variation/genetics , Mitogen-Activated Protein Kinase 1/genetics , Phosphorylation , Plasmids/genetics , Plasmids/metabolism , Substrate Specificity
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