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An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR

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Title: An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR
Authors: Sekiguchi, Mitsuhiro Browse this author
Kobashigawa, Yoshihiro Browse this author
Kawasaki, Masashi Browse this author
Yokochi, Masashi Browse this author
Kiso, Tetsuo Browse this author
Suzumura, Ken-ichi Browse this author
Mori, Keitaro Browse this author
Teramura, Toshio Browse this author
Inagaki, Fuyuhiko Browse this author →KAKEN DB
Keywords: DSC
FRB domain
fusion protein
Issue Date: Nov-2011
Publisher: Oxford University Press
Journal Title: Protein Engineering, Design and Selection
Volume: 24
Issue: 11
Start Page: 811
End Page: 817
Publisher DOI: 10.1093/protein/gzr045
Abstract: Mammalian target of rapamycin (mTOR), a large multi-domain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein-drug interactions at the domain level, while NMR gave insights into the protein-drug interactions at the residue level. The use of the FKBP12-FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.
Type: article
Appears in Collections:薬学研究院 (Faculty of Pharmaceutical Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 稲垣 冬彦

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