TY - JOUR
T1 - Development of a chemical screening system using aqueorin-fused protein
AU - Suzuki, Masafumi
AU - Furukawa, Shoichiro
AU - Kuramori, Chikanori
AU - Sawa, Chika
AU - Kabe, Yasuaki
AU - Nakamura, Mitsuhiro
AU - Sawada, Jun ichi
AU - Yamaguchi, Yuki
AU - Sakamoto, Satoshi
AU - Inouye, Satoshi
AU - Handa, Hiroshi
N1 - Funding Information:
We thank Mr. Kosuke Nishio and Mr. Mitsunori Omachi for preparation of materials. This work was supported by a Grant for Research and Development Projects in Cooperation with Academic Institutions from the New Energy and Industrial Technology Development Organization (NEDO) and by Special Coordination Funds for Promoting Science and Technology from the Japan Science and Technology Agency (JST).
PY - 2008/4/11
Y1 - 2008/4/11
N2 - 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.
AB - 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.
KW - Aequorin (AQ)
KW - Affinity purification
KW - Chemical screening system
KW - Fusion protein
KW - Magnetic nanobeads (FG beads)
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U2 - 10.1016/j.bbrc.2008.01.107
DO - 10.1016/j.bbrc.2008.01.107
M3 - Article
C2 - 18243129
AN - SCOPUS:39749175509
SN - 0006-291X
VL - 368
SP - 600
EP - 605
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -