Expression, purification, and inhibition of human RET tyrosine kinase

Luca Mologni, Elisa Sala, Barbara Riva, Luca Cesaro, Sara Cazzaniga, Sara Redaelli, Oriano Marin, Nicola Pasquato, Arianna Donella-Deana, Carlo Gambacorti-Passerini

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)177-185
Number of pages9
JournalProtein Expression and Purification
Volume41
Issue number1
DOIs
Publication statusPublished - May 2005

Keywords

  • ELISA
  • Immobilised metal affinity chromatography
  • Inhibitor screening
  • PTC
  • RET
  • Thyroid
  • Tyrosine kinase

ASJC Scopus subject areas

  • Biochemistry

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