Development of a chemical screening system using aqueorin-fused protein.
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.
Suzuki
Masafumi
M
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Furukawa
Shoichiro
S
Kuramori
Chikanori
C
Sawa
Chika
C
Kabe
Yasuaki
Y
Nakamura
Mitsuhiro
M
Sawada
Jun-Ichi
J
Yamaguchi
Yuki
Y
Sakamoto
Satoshi
S
Inouye
Satoshi
S
Handa
Hiroshi
H
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
D000331:Aequorin; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate