An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR
Mammalian target of rapamycin (mTOR), a large multidomain 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...
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| Veröffentlicht in: | Protein engineering, design and selection design and selection, 2011-11, Vol.24 (11), p.811-817 |
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| container_title | Protein engineering, design and selection |
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| creator | Sekiguchi, Mitsuhiro Kobashigawa, Yoshihiro Kawasaki, Masashi Yokochi, Masashi Kiso, Tetsuo Suzumura, Ken-ichi Mori, Keitaro Teramura, Toshio Inagaki, Fuyuhiko |
| description | Mammalian target of rapamycin (mTOR), a large multidomain 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. |
| doi_str_mv | 10.1093/protein/gzr045 |
| format | Article |
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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.</description><identifier>ISSN: 1741-0126</identifier><identifier>EISSN: 1741-0134</identifier><identifier>DOI: 10.1093/protein/gzr045</identifier><identifier>PMID: 21900305</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Calorimetry, Differential Scanning - methods ; Enzyme Inhibitors - chemistry ; Enzyme Inhibitors - pharmacology ; Escherichia coli ; Humans ; Ligands ; Magnetic Resonance Spectroscopy - methods ; Original ; Protein Binding - drug effects ; Protein Structure, Tertiary ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - metabolism ; Tacrolimus Binding Protein 1A - antagonists & inhibitors ; Tacrolimus Binding Protein 1A - chemistry ; Tacrolimus Binding Protein 1A - metabolism ; TOR Serine-Threonine Kinases - antagonists & inhibitors ; TOR Serine-Threonine Kinases - chemistry ; TOR Serine-Threonine Kinases - metabolism</subject><ispartof>Protein engineering, design and selection, 2011-11, Vol.24 (11), p.811-817</ispartof><rights>The Author 2011. 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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.</description><subject>Calorimetry, Differential Scanning - methods</subject><subject>Enzyme Inhibitors - chemistry</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Escherichia coli</subject><subject>Humans</subject><subject>Ligands</subject><subject>Magnetic Resonance Spectroscopy - methods</subject><subject>Original</subject><subject>Protein Binding - drug effects</subject><subject>Protein Structure, Tertiary</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Tacrolimus Binding Protein 1A - antagonists & inhibitors</subject><subject>Tacrolimus Binding Protein 1A - chemistry</subject><subject>Tacrolimus Binding Protein 1A - metabolism</subject><subject>TOR Serine-Threonine Kinases - antagonists & inhibitors</subject><subject>TOR Serine-Threonine Kinases - chemistry</subject><subject>TOR Serine-Threonine Kinases - metabolism</subject><issn>1741-0126</issn><issn>1741-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>TOX</sourceid><sourceid>EIF</sourceid><recordid>eNpVkUtPwzAMgCME4n3liHJDSJQ5bdLHBWkMBoinYJyjdElGUJuUPibBnf9N2MaAkyP702fHRmiPwDGBLOpVtWuVsb3JRw2UraBNklASAIno6vIdxhtoq2leAcI4IWQdbYQkA4iAbaLPvsVqKopOtMZZ3DpXYO1qPLw-fSBhIFWlrFS2xcJKHBj7myhH94_Y2BeTm9bVDe4aYydY4LErc2PnOqdnomDGak_43GLiI3z2NJhZ724fd9CaFkWjdhdxGz0Pz0eDy-Dm_uJq0L8JrB-2DTIhgQlKWSZpFMZSyzxPIJYpaKoUEJ8GlqZCk5hpGSnKfCQs9EiiYqqjbXQy91ZdXio59v-oRcGr2pSifudOGP6_Ys0Ln7gpj0gWpwl4wcFCULu3TjUtL00zVkUhrHJdwzOSAsvS8Jvc_9tq2eNn9R44nAOuq5ZVAvz7rHyxJD4_a_QFRWKWTg</recordid><startdate>20111101</startdate><enddate>20111101</enddate><creator>Sekiguchi, Mitsuhiro</creator><creator>Kobashigawa, Yoshihiro</creator><creator>Kawasaki, Masashi</creator><creator>Yokochi, Masashi</creator><creator>Kiso, Tetsuo</creator><creator>Suzumura, Ken-ichi</creator><creator>Mori, Keitaro</creator><creator>Teramura, Toshio</creator><creator>Inagaki, Fuyuhiko</creator><general>Oxford University Press</general><scope>TOX</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>5PM</scope></search><sort><creationdate>20111101</creationdate><title>An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR</title><author>Sekiguchi, Mitsuhiro ; Kobashigawa, Yoshihiro ; Kawasaki, Masashi ; Yokochi, Masashi ; Kiso, Tetsuo ; Suzumura, Ken-ichi ; Mori, Keitaro ; Teramura, Toshio ; Inagaki, Fuyuhiko</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-n305t-9ad05a4459d4326dfdbb706d80f4ee019d40588af165fd3e4565f15206d7e64f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Calorimetry, Differential Scanning - methods</topic><topic>Enzyme Inhibitors - chemistry</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Escherichia coli</topic><topic>Humans</topic><topic>Ligands</topic><topic>Magnetic Resonance Spectroscopy - methods</topic><topic>Original</topic><topic>Protein Binding - drug effects</topic><topic>Protein Structure, Tertiary</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Tacrolimus Binding Protein 1A - antagonists & inhibitors</topic><topic>Tacrolimus Binding Protein 1A - chemistry</topic><topic>Tacrolimus Binding Protein 1A - metabolism</topic><topic>TOR Serine-Threonine Kinases - antagonists & inhibitors</topic><topic>TOR Serine-Threonine Kinases - chemistry</topic><topic>TOR Serine-Threonine Kinases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sekiguchi, Mitsuhiro</creatorcontrib><creatorcontrib>Kobashigawa, Yoshihiro</creatorcontrib><creatorcontrib>Kawasaki, Masashi</creatorcontrib><creatorcontrib>Yokochi, Masashi</creatorcontrib><creatorcontrib>Kiso, Tetsuo</creatorcontrib><creatorcontrib>Suzumura, Ken-ichi</creatorcontrib><creatorcontrib>Mori, Keitaro</creatorcontrib><creatorcontrib>Teramura, Toshio</creatorcontrib><creatorcontrib>Inagaki, Fuyuhiko</creatorcontrib><collection>Oxford Journals Open Access Collection</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Protein engineering, design and selection</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sekiguchi, Mitsuhiro</au><au>Kobashigawa, Yoshihiro</au><au>Kawasaki, Masashi</au><au>Yokochi, Masashi</au><au>Kiso, Tetsuo</au><au>Suzumura, Ken-ichi</au><au>Mori, Keitaro</au><au>Teramura, Toshio</au><au>Inagaki, Fuyuhiko</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR</atitle><jtitle>Protein engineering, design and selection</jtitle><addtitle>Protein Eng Des Sel</addtitle><date>2011-11-01</date><risdate>2011</risdate><volume>24</volume><issue>11</issue><spage>811</spage><epage>817</epage><pages>811-817</pages><issn>1741-0126</issn><eissn>1741-0134</eissn><abstract>Mammalian target of rapamycin (mTOR), a large multidomain 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. 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| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Calorimetry, Differential Scanning - methods Enzyme Inhibitors - chemistry Enzyme Inhibitors - pharmacology Escherichia coli Humans Ligands Magnetic Resonance Spectroscopy - methods Original Protein Binding - drug effects Protein Structure, Tertiary Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - metabolism Tacrolimus Binding Protein 1A - antagonists & inhibitors Tacrolimus Binding Protein 1A - chemistry Tacrolimus Binding Protein 1A - metabolism TOR Serine-Threonine Kinases - antagonists & inhibitors TOR Serine-Threonine Kinases - chemistry TOR Serine-Threonine Kinases - metabolism |
| title | 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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