人Hepcidin融合表达载体的构建及在大肠杆菌中的表达

为了在大肠杆菌中表达生产hepcidin,根据大肠杆菌密码子偏好性,化学合成了人hepcidin的基因序列,并构建了hepcidin的融合表达载体pET -hpc。pET- hpc在大肠杆菌BL2 1 (DE3)中表达的hepcidin融合蛋白以包涵体形式存在,其N端带有 6个组氨酸。通过优化诱导表达条件,该融合蛋白表达水平显著提高,占总蛋白的 2 5 . 2 %。表达的包涵体经 1 %TritonX 1 0 0洗涤后溶于8mol L尿素,在变性条件下采用金属螯合层析进行纯化,所得融合蛋白纯度大于 95 %。

Construction of Human Hepcidin Expression Vector and Its Expression in Escherichia coli

Human hepcidin is a low-molecular-weight, cysteine-rich peptide relevant to small intestine iron absorption and body iron homeostasis. A DNA fragment containing the coding sequence of human hepcidin was designed according to codon preference of E. coli and chemically synthesized. The plasmid pET-hpc was constructed and His-tagged hepcidin was expressed in E. coli BL21(DE3). The fusion protein existed in the form of inclusion bodies. After induction conditions were optimized, the protein expression level was up to 25.2%. Following washing and dissolution of inclusion bodies, the fusion protein was purified by immobilized metal ion affinity chromatography under denaturation condition. The protein purity was >95%.