title: Expression and purification of a natural N-terminal pre-ligand assembly domain of tumor necrosis factor receptor 1 (TNFR1 PLAD) and preliminary activity determination.
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.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Cao
authors.forename: Fang
authors.initials: F
authors.lastname: Meng
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
authors.forename: Hongxia
authors.initials: H
authors.lastname: Dong
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004765:Enteropeptidase / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D008024:Ligands; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D005982:Glutathione Transferase; D004765:Enteropeptidase