可溶性三聚化CD40-异亮氨酸拉链融合蛋白的制备及鉴定

Preparation and Identification of Soluble Trimeric CD40-isoleucine Zipper Fusion Protein

以克隆的CD40cDNA为模板,经多步PCR构建羧基端融合异亮氨酸拉链(isoleucine zipper,IZ)三聚化基序和His6标签的可溶性CD40融合蛋白(sCD40IZ)的原核表达载体,在大肠杆菌中获得高效表达,分子量为27kD,与理论大小相符,表达产物主要存在于包涵体中,对包涵体蛋白进行稀释复性和纯化得到可溶性的sCD40IZ重组蛋白,该蛋白在溶液中的分子量为91kD,表明最有可能以三聚体形式存在。活性分析显示该蛋白能够与细胞上的CD40L结合,并且其结合活性与不含IZ基序的可溶性CD40相比明显提高。这些结果表明,在可溶性CD40羧基端融合IZ基序能够促进形成三聚体,并且具有增强的配基结合活性。

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.