title: Development of a chemical screening system using aqueorin-fused protein.
abstract: We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Furukawa
authors.forename: Chikanori
authors.initials: C
authors.lastname: Kuramori
authors.forename: Chika
authors.initials: C
authors.lastname: Sawa
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kabe
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Nakamura
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Sawada
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inouye
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Handa
mesh_terms: D000331:Aequorin / Q000032:analysis*; D016503:Drug Delivery Systems / Q000379:methods; D018189:Immunomagnetic Separation / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D008727:Methotrexate / Q000032:analysis* / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis* / Q000737:chemistry
chemical_list: D000331:Aequorin; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate