title: Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system.
abstract: Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.
authors.affiliation: Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111, USA.
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Maki
authors.forename: J
authors.initials: J
authors.lastname: Tres Brazell
authors.forename: Xin
authors.initials: X
authors.lastname: Teng
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Cuny
authors.forename: Alexei
authors.initials: A
authors.lastname: Degterev
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007093:Imidazoles; D007211:Indoles; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C507699:necrostatin-1; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases