| Expression, purification, and inhibition of human RET tyrosine kinase. | |
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MedLine Citation:
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PMID: 15802236 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay. |
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Authors:
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Luca Mologni; Elisa Sala; Barbara Riva; Luca Cesaro; Sara Cazzaniga; Sara Redaelli; Oriano Marin; Nicola Pasquato; Arianna Donella-Deana; Carlo Gambacorti-Passerini |
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Publication Detail:
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Type: In Vitro; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Protein expression and purification Volume: 41 ISSN: 1046-5928 ISO Abbreviation: Protein Expr. Purif. Publication Date: 2005 May |
Date Detail:
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Created Date: 2005-04-01 Completed Date: 2005-08-22 Revised Date: 2012-06-04 |
Medline Journal Info:
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Nlm Unique ID: 9101496 Medline TA: Protein Expr Purif Country: United States |
Other Details:
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Languages: eng Pagination: 177-85 Citation Subset: IM |
Affiliation:
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Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy. luca.mologni@unimib.it |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Baculoviridae / genetics Base Sequence Catalytic Domain Cell Line DNA, Recombinant / genetics Enzyme Inhibitors / chemistry, pharmacology Gene Expression Humans Molecular Sequence Data Molecular Structure Oncogene Proteins / antagonists & inhibitors, genetics*, isolation & purification* Proto-Oncogene Proteins c-ret Receptor Protein-Tyrosine Kinases / antagonists & inhibitors, genetics*, isolation & purification* Recombinant Proteins / antagonists & inhibitors, genetics, isolation & purification Spodoptera |
| Chemical | |
Reg. No./Substance:
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0/DNA, Recombinant; 0/Enzyme Inhibitors; 0/Oncogene Proteins; 0/Recombinant Proteins; EC 2.7.10.1/Proto-Oncogene Proteins c-ret; EC 2.7.10.1/RET protein, human; EC 2.7.10.1/Receptor Protein-Tyrosine Kinases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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