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Expression and Purification of a Natural N-Terminal Pre-ligand Assembly Domain of Tumor Necrosis Factor Receptor 1 (TNFR1 PLAD) and Preliminary Activity Determination

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Abstract

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-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.

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Abbreviations

TNFR:

Tumor necrosis factor receptor

PLAD:

Pre-ligand binding assembly domain

IMID:

Immune-mediated inflammatory diseases

GST:

Glutathione S-transferase

IPTG:

Lactose analog isopropyl β-D-thiogalactoside

PVDF:

Polyvinylidene difluoride membrane

MTT:

(4,4-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide

OPD:

o-Phenylenediamine dihydrochloride

LB:

Luria-Bertani

ELISA:

Enzyme-linked immunosorbent assay

DAB:

Diaminobenzidine

TFA:

Trifluoroacetic acid

CD:

Circular dichroism spectra

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Acknowledgments

This study was supported by grants from the Jiangsu Provincial Research Innovation Program for College Graduates (CX09B_290Z) and Qing Lan Project.

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Authors and Affiliations

  1. School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China

    Jin Cao, Fang Meng, Xiangdong Gao, Hongxia Dong & Wenbing Yao

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  1. Jin Cao
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  2. Fang Meng
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  3. Xiangdong Gao
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  4. Hongxia Dong
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  5. Wenbing Yao
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Correspondence to Wenbing Yao.

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Cao, J., Meng, F., Gao, X. et al. 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 30, 281–289 (2011). https://doi.org/10.1007/s10930-011-9330-4

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