Abstract
BackgroundCD40, 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.ResultsA 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.ConclusionsThe 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.
Highlights
CD40, called Bp50, is a novel member of the TNF receptor superfamily
Transformation of P. pastoris with pPIC9K yielded 4 transformants that were able to grow in the presence of a high concentration of geneticin, including three strains that grew in 4 mg/mL geneticin on YPD plates and one strain that grew in 1 mg/mL geneticin
Coomassie blue staining detected an increasing band at approximately 27 kDa that peaked at 96 h, suggesting that CD40-N may be secreted into the culture medium
Summary
CD40, 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. CD40 is a 50-kDa transmembrane protein that belongs to the TNF receptor family It is expressed on antigen-presenting cells such as B cells, dendritic cells and macrophages, but is found on endothelial cells, mast cells, fibroblast cells, tumor cells and smooth. In addition to the antigenpresenting process, CD40-activated signals are involved in the priming of T cells [12], the cytotoxicity of T cells [13], the proliferation and differentiation of B cells, and immunoglobulin class switching and so on [14] It participates in physiological processes, many studies have been published on its role in the pathology of disease. The CD40 signaling pathway is considered to be a promising target for the clinical treatment of autoimmune diseases [17,18,19]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
