Expression, Purification And Biological Characterization Of Extra-cellular Domain Of CD40 In Pichia Pastoris

CD40 also called Bp50 is a novel member of TNF-receptor superfamily and CD40L-CD40 signaling pathway is critical in immune system.Based on its important functions in improving antigen presentation function of professional antigen-presenting cells such as dendritic cells and activating B cells,CD40 signaling has become a vital target in treating transplantation rejection and autoimmune diseases as well as promoting anti-tumor immune response.In the present study,the cDNA coding for the extracellular domain of the CD40(CD40-N)has been optimized,cloned and expressed in organism Pichia pastoris.SDS-PAGE and Western blotting assay of culture broth from a methanol-induced expression strain revealed that recombinant CD40-N,a 27 kDa glycosylated protein,was secreted into culture medium.The recombinant protein was purified by using Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchangechromatography steps.Finally,67 mg of the protein was obtained in high purity from 3L of the supernatant.Bioactivity of recombinant CD40-N is confirmed by the ability of the protein to disrupt non-canonical NF-?B signal pathway in BJAB cells in vitro.Furthermore,a new function of CD40-N has been found as the protein can inhibit the growth of DLBCL tumor in vivo.Our result suggests that the P.pastoris expression system can be used to produce large quantities of fully functional CD40-N for both research and industrial purpose.