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目的:通过对大肠杆菌表达重组人骨形成蛋白-2的复性研究,得到高活性的重组人骨形成蛋白-2。方法:在大肠杆菌中通过温度诱导表达重组人骨形成蛋白-2经过Triton X-100清洗之后,又通过DEAE离子交换层析纯化包涵体,包涵体在8 mol/L尿素变性溶解,在氧化-还原(还原型和氧化型谷胱甘肽)复性系统中,通过简单的稀释复性,通过肝素亲和层析一步纯化法纯化重组人骨形成蛋白-2 ,最后通过诱导C2C12细胞产生碱性磷酸酶检测重组人骨形成蛋白-2活性。结果:温度诱导表达重组人骨形成蛋白-2是以包涵体单体的形式存在,经过几步纯化后,得到高纯度的包涵体。重组人骨形成蛋白-2在不同氧化-还原剂比例,和不同小分子化学辅助剂浓度中复性,得到复性的效率也不同。亲和层析纯化后,本实验得到重组人骨形成蛋白-2的生物学活性比商业化的重组人骨形成蛋白-2更高。结论:重组人骨形成蛋白-2属于转化因子-β家族,此复性方法可能应用于此家族的其他成员同时得到成本低、产量高的重组人骨形成蛋白-2 ,可能为临床应用创造了良好的条件。
OBJECTIVE: To study the renaturation of recombinant human bone morphogenetic protein-2 in Escherichia coli to obtain highly active recombinant human bone morphogenetic protein-2. Methods: Recombinant human bone morphogenetic protein-2 was induced to express in E.coli by Triton X-100 and purified by DEAE ion exchange chromatography. The inclusion bodies were denatured and dissolved in 8 mol / L urea, (Reduced and oxidized glutathione) refolding system, recombinant human bone morphogenetic protein-2 was purified by one-step purification of heparin affinity chromatography by simple dilution refolding, and finally, by inducing C2C12 cells to produce alkaline phosphatase Recombinant human bone morphogenetic protein-2 activity was measured. Results: Temperature-induced expression of recombinant human bone morphogenetic protein-2 was in the form of inclusion body monomer. After several steps of purification, high purity inclusion body was obtained. Recombinant human bone morphogenetic protein-2 refolding efficiency in different oxidant - reductant concentration, and different small molecule chemical adjuvant concentration refolding. After purification by affinity chromatography, we found that the biological activity of recombinant human bone morphogenetic protein-2 was higher than that of commercial recombinant human bone morphogenetic protein-2. Conclusion: Recombinant human bone morphogenetic protein-2 belongs to the transforming factor-β family. This refolding method may be applied to other members of this family to obtain low-cost and high-yield recombinant human bone morphogenetic protein-2 at the same time, which may create a good clinical application condition.