[Preparation and identification of soluble trimeric CD40-isoleucine zipper fusion protein].
The interaction of CD40 with its cognate ligand, CD40L (CD154), plays important roles in immune responses. Blockade of CD40-CD40L signal pathway can protect the progression of antibody- and cell-mediated autoimmune diseases, and reduce allograft rejection thus prolonging graft survival, even engendering long-lived antigen-specific tolerance. The present study aims to enhance the binding activity of CD40 by incorporating an isoleucine zipper (IZ) trimeric motif into CD40 ectodomain to promote the formation of soluble CD40 trimers, which would be useful for blocking CD40-CD40L interaction. A prokaryotic expression vector for soluble human CD40 ectodomain fused with an IZ motif and a hexa-histidine (His6) tag at its carboxyl terminus (sCD40IZ) was constructed by multiple round PCR using cloned CD40 cDNA as a template. The recombinant sCD40IZ protein was expressed highly in Escherichia coli (E. coli) with a molecular weight of 27kD, which is consistent with its theoretical value. It mainly existed in inclusion bodies. After refolding from inclusion bodies, soluble sCD40IZ protein was purified by gel filtration. Its molecular weight in solution was about 91kD when determined by gel filtration, suggesting that it most probably existed in the form of trimers. Moreover, this protein could bind to CD40L expressed on Jurkat T cells and its binding activity was significantly higher than that of soluble CD40 without an IZ motif. These results suggest that incorporation of an IZ motif at the carboxyl terminus of soluble CD40 can facilitate the formation of trimers and enhance its binding activity with CD40L. Thus, the trimeric CD40 protein may be used to block CD40-CD40L signal pathway, suggesting that it may have potential application in preventing autoimmune diseases and transplantation rejection.
Xu
Li-Hui
LH
Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China.
Liu
Yi
Y
He
Xian-Hui
XH
D019013:CD40 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000096:biosynthesis* / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D019013:CD40 Antigens; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D023201:CD40 Ligand