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

Sekiguchi, Mitsuhiro; Kobashigawa, Yoshihiro; Kawasaki, Masashi; Yokochi, Masashi; Kiso, Tetsuo; Suzumura, Ken-ichi; Mori, Keitaro; Teramura, Toshio; Inagaki, Fuyuhiko

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

Sekiguchi, Mitsuhiro; Kobashigawa, Yoshihiro; Kawasaki, Masashi et al.

Protein Engineering, Design and Selection, 2011, 24(11), 811-817

Permalink : https://hdl.handle.net/2115/47434

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アクセス権	open access
URI	https://hdl.handle.net/2115/47434
タイトル	An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR
著者	Sekiguchi, Mitsuhiro ; Kobashigawa, Yoshihiro ; Kawasaki, Masashi ; Yokochi, Masashi ; Kiso, Tetsuo ; Suzumura, Ken-ichi ; Mori, Keitaro ; Teramura, Toshio ; Inagaki, Fuyuhiko
著者名 Sekiguchi, Mitsuhiro
著者名 Kobashigawa, Yoshihiro
著者名 Kawasaki, Masashi
著者名 Yokochi, Masashi
著者名 Kiso, Tetsuo
著者名 Suzumura, Ken-ichi
著者名 Mori, Keitaro
著者名 Teramura, Toshio
e-Rad_Researcher 70011757 著者名 Inagaki, Fuyuhiko
言語	英語
キーワード	DSC
	FRB domain
	NMR
	fusion protein
発行日	2011-11
出版者	Oxford University Press
収録物名	Protein Engineering, Design and Selection
巻(号)	24(11)
ページ	811-817
抄録	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.
資源タイプ	学術雑誌論文
出版タイプ	Version of Record
PISSN	1741-0126
関連情報 (isIdenticalTo)	出版社版DOI https://doi.org/10.1093/protein/gzr045