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Published on: August 17, 2019
High-throughput screening assay for D-amino acid oxidase
Svetlana V Khoronenkova1, Vladimir I Tishkov
1Department of Chemical Enzymology, Chemistry Faculty, M. V. Lomonosov Moscow State University, 119992 Moscow, Russia.
Analytical Biochemistry
|January 15, 2008
Summary
A new high-throughput screening (HTS) assay detects active D-amino acid oxidase (DAAO) in samples and E. coli cells. This method offers sensitive and quantitative enzyme detection for screening mutant DAAOs.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- D-amino acid oxidase (DAAO) plays a role in various biological processes and has therapeutic potential.
- Developing efficient methods for detecting and quantifying DAAO activity is crucial for research and drug discovery.
- Existing assays may have limitations in throughput, sensitivity, or applicability to intact cells.
Purpose of the Study:
- To develop and optimize a novel membrane-based high-throughput screening (HTS) assay for DAAO activity.
- To enable the detection and quantification of DAAO in both liquid samples and intact Escherichia coli cells.
- To validate the assay's utility in screening mutant DAAOs for activity against target substrates.
Main Methods:
- Development of a membrane-based HTS assay utilizing specific detection principles for DAAO.
- Optimization of assay conditions for sensitivity and quantitative performance.
- Application of the assay to screen a library of E. coli clones expressing mutant DAAOs.
Main Results:
- The developed HTS assay demonstrated a detection limit of less than 1 ng per sample.
- The assay provides quantitative DAAO determination within the range of 0.13 to 3.60 ng enzyme per probe.
- Successful screening of a library of E. coli clones containing mutant DAAOs active toward target substrates was achieved.
Conclusions:
- A robust and sensitive membrane-based HTS assay for DAAO has been successfully developed and optimized.
- The assay is suitable for both liquid samples and intact E. coli cells, offering versatility.
- This method facilitates efficient screening of mutant DAAOs, advancing research in enzymology and potential therapeutic applications.
