High-throughput screening assay for D-amino acid oxidase.
A membrane-based high-throughput screening (HTS) assay for active D-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.
Khoronenkova
Svetlana V
SV
Department of Chemical Enzymology, Chemistry Faculty, M. V. Lomonosov Moscow State University, 119992 Moscow, Russia.
Tishkov
Vladimir I
VI
D000594:Amino Acid Oxidoreductases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D003101:Collodion / Q000737:chemistry; D004338:Drug Combinations; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D005048:Etorphine / Q000737:chemistry; D008567:Membranes, Artificial; D008728:Methotrimeprazine / Q000737:chemistry; D016296:Mutagenesis; D009154:Mutation; D015203:Reproducibility of Results
D000596:Amino Acids; D004338:Drug Combinations; D008567:Membranes, Artificial; C004820:Immobilon; D005048:Etorphine; D003101:Collodion; D008728:Methotrimeprazine; D000594:Amino Acid Oxidoreductases