title: Expression, purification, and inhibition of human RET tyrosine kinase.
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.
authors.affiliation: Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy. luca.mologni@unimib.it
authors.forename: Luca
authors.initials: L
authors.lastname: Mologni
authors.forename: Elisa
authors.initials: E
authors.lastname: Sala
authors.forename: Barbara
authors.initials: B
authors.lastname: Riva
authors.forename: Luca
authors.initials: L
authors.lastname: Cesaro
authors.forename: Sara
authors.initials: S
authors.lastname: Cazzaniga
authors.forename: Sara
authors.initials: S
authors.lastname: Redaelli
authors.forename: Oriano
authors.initials: O
authors.lastname: Marin
authors.forename: Nicola
authors.initials: N
authors.lastname: Pasquato
authors.forename: Arianna
authors.initials: A
authors.lastname: Donella-Deana
authors.forename: Carlo
authors.initials: C
authors.lastname: Gambacorti-Passerini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera
chemical_list: D004274:DNA, Recombinant; D004791:Enzyme Inhibitors; D015513:Oncogene Proteins; D011994:Recombinant Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases