Characterization of a soluble IL-6 receptor alpha mutant C277D/H280I expressed in E.coli

Biochem Mol Biol Int. 1997 May;41(6):1101-8. doi: 10.1080/15216549700202181.

Abstract

The pleiotropic cytokine interleukin-6(IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal transducer gp130. To construct the antagonist of IL-6, a DNA segment encoding the soluble human IL-6R alpha (shIL-6R alpha) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction. The DNA segment was then subcloned into vector pET-3b and expressed in E.coli at a high level. The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL-6-binding capability by 8-fold. DM650 antagonized IL-6 perfectly, resulting in the growth-inhibition of human myeloma AF-10 cells. DNA fragmentation assay proved that the growth of AF-10 cells was inhibited through the induction of apoptosis suppressed by IL-6.

MeSH terms

  • Antigens, CD / chemistry*
  • Antigens, CD / genetics*
  • Antigens, CD / metabolism
  • Apoptosis
  • Escherichia coli / chemistry
  • Escherichia coli / genetics*
  • Genetic Vectors / metabolism
  • Humans
  • Interleukin-6 / antagonists & inhibitors
  • Interleukin-6 / metabolism*
  • Mutagenesis, Site-Directed*
  • Protein Binding
  • Protein Folding
  • Receptors, Interleukin / chemistry*
  • Receptors, Interleukin / genetics*
  • Receptors, Interleukin / metabolism
  • Receptors, Interleukin-6
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Solubility
  • Structure-Activity Relationship
  • Tumor Cells, Cultured

Substances

  • Antigens, CD
  • Interleukin-6
  • Receptors, Interleukin
  • Receptors, Interleukin-6
  • Recombinant Proteins