Expression, purification and biological characterization of the extracellular domain of CD40 from Pichia pastoris

BMC Biotechnology
Yu ZhanXiaoren Zhang

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-...Continue Reading

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Methods Mentioned

BETA
size-exclusion
ion exchange chromatography
restriction digest
PCR
ion-exchange chromatography
glycosylation

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