Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system

Protein Expr Purif. 2013 Jun;89(2):156-61. doi: 10.1016/j.pep.2013.03.002. Epub 2013 Mar 21.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Baculoviridae / genetics*
  • Cell Line
  • Cloning, Molecular*
  • Humans
  • Imidazoles / pharmacology
  • Indoles / pharmacology
  • Protein Kinase Inhibitors / pharmacology
  • Receptor-Interacting Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Receptor-Interacting Protein Serine-Threonine Kinases / genetics*
  • Receptor-Interacting Protein Serine-Threonine Kinases / isolation & purification
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Imidazoles
  • Indoles
  • Protein Kinase Inhibitors
  • Recombinant Proteins
  • necrostatin-1
  • RIPK1 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases