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• W2272631627 abstract "CD40, also called Bp50, is a novel member of the TNF receptor superfamily. Based on its important role in multiple physiological and pathological processes, the CD40 signaling pathway has become a vital target for treating transplantation, autoimmune diseases and cancers. This study generated a protein fragment that disrupts this signaling pathway. A DNA fragment encoding the extracellular domain of CD40 (CD40-N) has been codon-optimized and cloned into pPIC9K to create a Pichia pastoris expression and secretion strain. SDS-PAGE and Western blotting assays using the culture media from methanol-induced expression strains showed that recombinant CD40-N, a 27 kDa glycosylated protein, was secreted into the culture broth. The recombinant protein was purified to more than 90 % using Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchange. Finally, 120 mg of the protein was obtained at a relatively high purity from 3 l supernatant. Binding assay (ITC200 assay) shown the direct interaction of CD40-N and CD40 agonist antibody (G28-5). The bioactivity of recombinant CD40-N was confirmed by its ability to disrupt non-canonical NF-κB signaling activated by CD40 agonist antibody or CD40 ligand and to inhibit ant-CD40 agonist antibody-induced TNF-alpha expression in BJAB cells in vitro. In addition, our data indicate that the protein has curative potential in treating dextran sulfate sodium (DSS)-induced colitis in vivo. The results show that the experimental procedure we have developed using P. pastoris can be used to produce large amounts of active CD40-N for research and industrial purposes. The protein fragment we have acquired has potential to be used in research or even treating inflammation diseases such as colitis." @default.
• W2272631627 created "2016-06-24" @default.
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• W2272631627 date "2016-01-25" @default.
• W2272631627 modified "2023-10-08" @default.
• W2272631627 title "Expression, purification and biological characterization of the extracellular domain of CD40 from Pichia pastoris" @default.
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• W2272631627 doi "https://doi.org/10.1186/s12896-016-0237-1" @default.
• W2272631627 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4727258" @default.
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• W2272631627 hasPublicationYear "2016" @default.
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