Journal
PROTEIN JOURNAL
Volume 30, Issue 4, Pages 281-289Publisher
SPRINGER
DOI: 10.1007/s10930-011-9330-4
Keywords
Tumor necrosis factor receptor (TNFR); Pre-ligand binding assembly domain (PLAD); GST fusion protein; L929 cells
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Funding
- Jiangsu Provincial Research Innovation Program for College Graduates [CX09B_290Z]
- Qing Lan Project
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A domain at the NH2 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-TNF alpha activity and the specific TNFR1 affinity were determined. The results proved that the natural N-terminal TNFR1 PLAD can selectively inhibit TNF alpha 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.
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