Work

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

Year: 2013

Type: book-chapter

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.

Source: Methods in molecular biology

DOI: 10.1007/978-1-62703-293-3_3

Authors: Olga Paley, Giulia Agnello, Jason Cantor, Tae Hyun Yoo, George Georgiou, Everett Stone

Institutions: The University of Texas at Austin, Ajou University

Language: English

Topic: Cancer Research and Treatments

Subfield: Biotechnology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funder: National Cancer Institute

Award: Engineering and Evaluation of Human L-Methionase for Cancer Therapy