DPP-rhBMP-4m融合表达载体的构建和应用

目的:构建容易去除标签蛋白的大肠杆菌融合蛋白表达载体.方法:PCR方法获得带有Asp-Pro-Pro前缀的重组人骨形态发生蛋白-4成熟肽(recombination human bone morphogenetic protein 4 mature peptide,rhBMP-4m)编码序列并克隆入pRSET载体,转化大肠杆菌后诱导表达;经免疫印迹法鉴定融合蛋白;甲酸裂解去除6His标签后细胞学实验检测其活性.结果:构建的融合表达载体序列测定结果与预期结果一致;诱导表达的DPP-rhBMP-4m融合蛋白以包涵体形式存在,占菌体总蛋白量的37%,亲和层析后目的蛋白纯度为97%;免疫印迹法鉴定显示,该融合蛋白可与相应抗血清产生特异性反应;对C2C12细胞具有良好的生物活性.结论:成功构建了利于目的蛋白纯化的pRSET-DPP-rhBMP-4m融合表达载体.

Construction and application of DPP-rhBMP-4m fusion protein expression vector

AIM: To construct a E.coli fusion protein expression vector from which tag protein can be removed easily. METHODS: The codons of Asp-Pro-Pro were added to 5'-teminal of rhBMP-4m (recombination human bone morphogenetic protein 4 mature peptide)gene by PCR. The DPP-rhBMP-4m coding sequence was cloned into E.coli BL21 and induced to express with IPTG. The expressed fusion protein (DPP-rhBMP-4m) was analyzed by SDS-PAGE and Western Blot. After the 6His tag protein was removed by formic acid, the purified PP-rhBMP-4m was added to C2C12 cells to observe its biological activity. RESULTS: The DNA sequencing showed that the sequence of the fusion protein expression vector constructed in this study was exactly consistent with the one predicted. The fusion protein occupied 37% of the total bacteria protein, while the purity of the target protein(rhBMP-4m) reached 97% after affinity chromatography. And the protein could specifically react with antiserum and showed a good biological activity in C2C12 cells. CONCLUSION: The pRSET-DPP-rhBMP-4m vector is successfully constructed for the purification of interest protein.