High Level Expression Of Recombinant Human IZ-CD40L And Identification Of Its Biological Activation

CD40 ligand(CD40L) plays a pivitol role in T-dependent humoral and cell-mediated immune responses by binding with its CD40 receptor, In this study, the isoleucine zipper(IZ) gene was fused at N-terminal of CD40L gene coding E107-L261 contributing to the formation of trimer. And the gene coding hexahistidine was fused at the N-terminal of IZ, so the IZ-CD40L fusion protein could be purified by affinity chromatography. The fusion gene was amplified and cloned into expression plasmid pET30a. The protein IZ-CD40L with His-tag at the N- terminal was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to acquire soluble IZ-CD40L. The fusion protein IZ-CD40L was conveniently purified using HiTrap~? affinity column with above 95% purity. The gel filtration chromatography and non-reduced SDS-PAGE identified the trimeric structure of the recombinant protein. The microscope analysis showed the IZ-CD40L could interact with the membrane CD40 on XG2, a multiple myeloma cell line. Furthermore, the in vitro bioassays demonstrated that IZ-CD40L could effectively promote the DC mature including augmenting the expression of CD83 CD86 HLA-DR and IL-12 secretion, and capturing FITC-dextran antigen significantly reduced.