Expression and purification of a natural N-terminal pre-ligand assembly domain of tumor necrosis factor receptor 1 (TNFR1 PLAD) and preliminary activity determination

Protein J. 2011 Apr;30(4):281-9. doi: 10.1007/s10930-011-9330-4.

Abstract

A domain at the NH(2) terminal (N-terminal) of tumor necrosis factor receptor (TNFR) termed the pre-ligand binding assembly domain (PLAD). The finding that PLAD can mediate a selective TNFR assembly in previously researches provides a novel target to the prevention of TNFR signaling in immune-mediated inflammatory diseases (IMID). In this study, a natural N-terminal TNFR1 PLAD was obtained for the first time through the methods of GST-tag fusion protein expression and enterokinase cleavage. After purification with a Q Sepharose Fast Flow column, a natural N-terminal TNFR1 PLAD which purity was up to 95%, was obtained and was identified using Nano LC-ECI-MS/MS. Secondary structure analysis of PLAD was carried out using circular dichroism spectra (CD). After that, the TNFR1 PLAD in vitro anti-TNFα activity and the specific TNFR1 affinity were determined. The results proved that the natural N-terminal TNFR1 PLAD can selectively inhibit TNFα bioactivity mainly through TNFR1. It infers an effective and safe strategy for treating variety of IMID with a low risk of side effects in future.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Line
  • Enteropeptidase / metabolism
  • Gene Expression
  • Glutathione Transferase / genetics*
  • Humans
  • Ligands
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Plasmids / genetics
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptors, Tumor Necrosis Factor, Type I / chemistry*
  • Receptors, Tumor Necrosis Factor, Type I / genetics*
  • Receptors, Tumor Necrosis Factor, Type I / isolation & purification
  • Receptors, Tumor Necrosis Factor, Type I / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Ligands
  • Receptors, Tumor Necrosis Factor, Type I
  • Recombinant Fusion Proteins
  • Tumor Necrosis Factor-alpha
  • Glutathione Transferase
  • Enteropeptidase