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GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria

 Last Update: 2020-12-10
 Source: Internet
 Author: User

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complete amino acid

catalytic activity

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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 aminoacid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporterto 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 bacterialand human asparaginases and also for the catalytic optimization of an engineered human methionine gamma lyase.

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