Your browser doesn't support javascript.

Portal Regional da BVS

Informação e Conhecimento para a Saúde

Home > Pesquisa > ()
Imprimir Exportar

Formato de exportação:


Adicionar mais destinatários
| |

Recombinant expression, purification and bioactivity characterization of extracellular domain of human tumor necrosis factor receptor 1.

Protein Expr Purif; 155: 21-26, 2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30414969
The interaction between TNF-α with TNFR1 triggers important signaling pathways inducing diverse cellular phenomena including inflammation, apoptosis, etc., and is involved in the pathogenesis and progression of numerous autoimmune diseases. The extracellular domain (ECD) of TNFR has been successfully used to clinically treat such TNF-associated diseases. However, large-scale production of these biological material via eukaryotic cell expression systems is usually costly owing to the culture medium and complicated growth conditions. This study aimed to extract pure soluble human TNFR1-ECD and investigate its biological activity, using a prokaryotic expression system. Recombinant vector pMCSG7-TNFR1-ECD was constructed via ligation-independent cloning. The His-tag fusion protein was expressed in E. coli and localized in inclusion bodies. Recombinant TNFR1-ECD was refolded and purified via nickel-affinity chromatography, tag cleavage, followed by cation-exchange chromatography or size-exclusion chromatography. A purity of over 95% and a yield of 9.3 mg protein per liter of bacterial culture media was obtained. The purified protein showed significant affinity of 2.15 nM towards human TNF-α and inhibited TNF-α-mediated cytotoxicity in L929 cells, with an ED of 0.10 µg/ml. It formed a self-associated oligomer with a K of 1.15 µM, detected via microscale thermophoresis. We thus established a highly efficient approach to construct, express, and purify the recombinant protein of human TNFR1-ECD from a prokaryotic system. The antagonistic bioactivities in vitro indicate this protein as a prospective molecules for drug research against autoimmune diseases characterized by TNF-α overexpression.