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Protein Expression & Purification, 2005, 41, 1, 177-185.
Mologni L, Sala E, Riva B, Cesaro L, Cazzaniga S, Redaelli S, Marin O,
Pasquato N, Donella-Deana A, Gambacorti-Passerini C.
Keywords: Immobilised metal affinity chromatography; Thyroid; RET;
PTC; Tyrosine kinase; Inhibitor screening; ELISA
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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