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. © 2005 Elsevier Inc. All rights reserved.

Mologni, L., Sala, E., Riva, B., Cesaro, L., Cazzaniga, S., Redaelli, S., et al. (2005). Expression, purification, and inhibition of human RET tyrosine kinase. PROTEIN EXPRESSION AND PURIFICATION, 41, 177-185 [10.1016/j.pep.2005.01.002].

Expression, purification, and inhibition of human RET tyrosine kinase

MOLOGNI, LUCA;REDAELLI, SARA;GAMBACORTI PASSERINI, CARLO
2005

Abstract

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. © 2005 Elsevier Inc. All rights reserved.
Articolo in rivista - Articolo scientifico
Scientifica
RET, tyrosine kinase
English
Mologni, L., Sala, E., Riva, B., Cesaro, L., Cazzaniga, S., Redaelli, S., et al. (2005). Expression, purification, and inhibition of human RET tyrosine kinase. PROTEIN EXPRESSION AND PURIFICATION, 41, 177-185 [10.1016/j.pep.2005.01.002].
Mologni, L; Sala, E; Riva, B; Cesaro, L; Cazzaniga, S; Redaelli, S; Marin, O; Pasquato, N; Donella Deana, A; GAMBACORTI PASSERINI, C
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/14686
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