Development and application of fully functional epitope-tagged forms of transforming growth factor-β
Administration of transforming growth factor-β (TGF-β) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricte...
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| Veröffentlicht in: | Journal of immunological methods 2002-08, Vol.266 (1), p.7-18 |
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| creator | Wolfraim, Lawrence A Alkemade, Gonnie M Alex, Biju Sharpe, Shellyann Parks, W.Tony Letterio, John J |
| description | Administration of transforming growth factor-β (TGF-β) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-β and related TGF-β isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-β1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-β signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-β1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-β and demonstrate the utility of these tagged ligands as probes for TGF-β receptor expression by flow cytometry. The design of these fully functional epitope-tagged TGF-β proteins should facilitate studies such as the evaluation of in vivo peptide pharmacodynamics and trafficking of TGF-β ligand–receptor complexes. |
| doi_str_mv | 10.1016/S0022-1759(02)00090-X |
| format | Article |
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However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-β and related TGF-β isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-β1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-β signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-β1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-β and demonstrate the utility of these tagged ligands as probes for TGF-β receptor expression by flow cytometry. The design of these fully functional epitope-tagged TGF-β proteins should facilitate studies such as the evaluation of in vivo peptide pharmacodynamics and trafficking of TGF-β ligand–receptor complexes.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/S0022-1759(02)00090-X</identifier><identifier>PMID: 12133618</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Amino Acid Sequence ; Animals ; Base Sequence ; Biological activity ; Biological and medical sciences ; Cell Line ; COS Cells ; Enzyme-Linked Immunosorbent Assay - methods ; Epitope tag ; Epitopes - analysis ; Flow Cytometry - methods ; Fundamental and applied biological sciences. 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However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-β and related TGF-β isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-β1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-β signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-β1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-β and demonstrate the utility of these tagged ligands as probes for TGF-β receptor expression by flow cytometry. The design of these fully functional epitope-tagged TGF-β proteins should facilitate studies such as the evaluation of in vivo peptide pharmacodynamics and trafficking of TGF-β ligand–receptor complexes.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological activity</subject><subject>Biological and medical sciences</subject><subject>Cell Line</subject><subject>COS Cells</subject><subject>Enzyme-Linked Immunosorbent Assay - methods</subject><subject>Epitope tag</subject><subject>Epitopes - analysis</subject><subject>Flow Cytometry - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>Hemagglutinins - genetics</subject><subject>Hemagglutinins - immunology</subject><subject>Microscopy, Confocal - methods</subject><subject>Microscopy, Fluorescence - methods</subject><subject>Molecular immunology</subject><subject>Molecular Sequence Data</subject><subject>Oligopeptides</subject><subject>Peptides - genetics</subject><subject>Peptides - immunology</subject><subject>Receptors, Cytoplasmic and Nuclear - analysis</subject><subject>Receptors, Transforming Growth Factor beta - analysis</subject><subject>Recombinant Fusion Proteins - analysis</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - pharmacology</subject><subject>Recombinant Proteins</subject><subject>Techniques</subject><subject>TGF-β</subject><subject>Transfection</subject><subject>Transforming Growth Factor beta - analysis</subject><subject>Transforming Growth Factor beta - chemistry</subject><subject>Transforming Growth Factor beta - genetics</subject><subject>Transforming Growth Factor beta - pharmacology</subject><subject>Transforming Growth Factor beta - physiology</subject><subject>Transforming Growth Factor beta1</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkM1u1DAQgK0K1G4Lj1CUSxEcAjNxnJ8TQqX8SJV6AKTeLK8z3ho5cWp7i_paPAjPVKe7okcuM5rRNz_6GDtFeIeAzfvvAFVVYiv6N1C9BYAeyusDtsKurcq2B_GMrf4hR-w4xl8ZQmjgkB1hhZw32K0YfaI7cn4eaUqFmoZCzbOzWiXrp8Kbwmydu89x0ktHuYJmm_xMZVKbDQ2F8WGMC5iCmuJS2WlTbIL_nW4Ko3Tyofz75wV7bpSL9HKfT9jPzxc_zr-Wl1dfvp1_vCw1F3Uq604AJ92vSXHEnut23Q-NUrzlgxmgrWvk1GihoDYNik5QJXqqG50DB6z4CXu92zsHf7ulmORooybn1ER-GyV2tYAWMYNiB-rgYwxk5BzsqMK9RJCLX_noVy7yJFTy0a-8znOv9ge265GGp6m90Ayc7QEVtXImW9E2PnG8g5bXfeY-7DjKOu4sBRm1pUnTYAPpJAdv__PKA1vUmQI</recordid><startdate>20020801</startdate><enddate>20020801</enddate><creator>Wolfraim, Lawrence A</creator><creator>Alkemade, Gonnie M</creator><creator>Alex, Biju</creator><creator>Sharpe, Shellyann</creator><creator>Parks, W.Tony</creator><creator>Letterio, John J</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T5</scope><scope>H94</scope></search><sort><creationdate>20020801</creationdate><title>Development and application of fully functional epitope-tagged forms of transforming growth factor-β</title><author>Wolfraim, Lawrence A ; Alkemade, Gonnie M ; Alex, Biju ; Sharpe, Shellyann ; Parks, W.Tony ; Letterio, John J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c354t-48503ec9bea31193c7b9d6aa373dfd074413e6c5a04f61585e259e46c9e430123</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Biological activity</topic><topic>Biological and medical sciences</topic><topic>Cell Line</topic><topic>COS Cells</topic><topic>Enzyme-Linked Immunosorbent Assay - methods</topic><topic>Epitope tag</topic><topic>Epitopes - analysis</topic><topic>Flow Cytometry - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Hemagglutinins - genetics</topic><topic>Hemagglutinins - immunology</topic><topic>Microscopy, Confocal - methods</topic><topic>Microscopy, Fluorescence - methods</topic><topic>Molecular immunology</topic><topic>Molecular Sequence Data</topic><topic>Oligopeptides</topic><topic>Peptides - genetics</topic><topic>Peptides - immunology</topic><topic>Receptors, Cytoplasmic and Nuclear - analysis</topic><topic>Receptors, Transforming Growth Factor beta - analysis</topic><topic>Recombinant Fusion Proteins - analysis</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - pharmacology</topic><topic>Recombinant Proteins</topic><topic>Techniques</topic><topic>TGF-β</topic><topic>Transfection</topic><topic>Transforming Growth Factor beta - analysis</topic><topic>Transforming Growth Factor beta - chemistry</topic><topic>Transforming Growth Factor beta - genetics</topic><topic>Transforming Growth Factor beta - pharmacology</topic><topic>Transforming Growth Factor beta - physiology</topic><topic>Transforming Growth Factor beta1</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wolfraim, Lawrence A</creatorcontrib><creatorcontrib>Alkemade, Gonnie M</creatorcontrib><creatorcontrib>Alex, Biju</creatorcontrib><creatorcontrib>Sharpe, Shellyann</creatorcontrib><creatorcontrib>Parks, W.Tony</creatorcontrib><creatorcontrib>Letterio, John J</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wolfraim, Lawrence A</au><au>Alkemade, Gonnie M</au><au>Alex, Biju</au><au>Sharpe, Shellyann</au><au>Parks, W.Tony</au><au>Letterio, John J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and application of fully functional epitope-tagged forms of transforming growth factor-β</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2002-08-01</date><risdate>2002</risdate><volume>266</volume><issue>1</issue><spage>7</spage><epage>18</epage><pages>7-18</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>Administration of transforming growth factor-β (TGF-β) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-β and related TGF-β isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-β1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-β signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-β1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-β and demonstrate the utility of these tagged ligands as probes for TGF-β receptor expression by flow cytometry. 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| subjects | Amino Acid Sequence Animals Base Sequence Biological activity Biological and medical sciences Cell Line COS Cells Enzyme-Linked Immunosorbent Assay - methods Epitope tag Epitopes - analysis Flow Cytometry - methods Fundamental and applied biological sciences. Psychology Fundamental immunology Hemagglutinins - genetics Hemagglutinins - immunology Microscopy, Confocal - methods Microscopy, Fluorescence - methods Molecular immunology Molecular Sequence Data Oligopeptides Peptides - genetics Peptides - immunology Receptors, Cytoplasmic and Nuclear - analysis Receptors, Transforming Growth Factor beta - analysis Recombinant Fusion Proteins - analysis Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - pharmacology Recombinant Proteins Techniques TGF-β Transfection Transforming Growth Factor beta - analysis Transforming Growth Factor beta - chemistry Transforming Growth Factor beta - genetics Transforming Growth Factor beta - pharmacology Transforming Growth Factor beta - physiology Transforming Growth Factor beta1 |
| title | Development and application of fully functional epitope-tagged forms of transforming growth factor-β |
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