Expression and Purification of JAK1 and SOCS1 for Structural and Biochemical Studies

Methods Mol Biol. 2018:1725:267-280. doi: 10.1007/978-1-4939-7568-6_21.

Abstract

Interferon gamma (IFNγ) is a potent inflammatory and immune cytokine. IFNγ signals via the interferon gamma receptor (IFNGR), which is constitutively bound to Janus Kinase (JAK) 1 and JAK2 via its intracellular domain. These two JAK proteins then initiate the inflammatory signaling cascade. The most potent inhibitor of IFNγ signaling is Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 negatively regulates IFNγ signaling pathway (and other pathways) by directly inhibiting JAKs. Here, we describe a protocol for the recombinant production and purification of the JAK1 kinase domain and its inhibitor SOCS1, for structural and biochemical studies.

Keywords: Janus Kinase (JAK); Protein expression; Protein purification; Suppressor of cytokine signaling (SOCS).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Baculoviridae / metabolism
  • Cells, Cultured
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Janus Kinase 1 / genetics
  • Janus Kinase 1 / isolation & purification*
  • Janus Kinase 1 / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sf9 Cells
  • Signal Transduction
  • Suppressor of Cytokine Signaling 1 Protein / genetics
  • Suppressor of Cytokine Signaling 1 Protein / isolation & purification*
  • Suppressor of Cytokine Signaling 1 Protein / metabolism*

Substances

  • Recombinant Proteins
  • SOCS1 protein, human
  • Suppressor of Cytokine Signaling 1 Protein
  • JAK1 protein, human
  • Janus Kinase 1