重组人锰超氧化物歧化酶纯化条件的研究.docVIP

重组人锰超氧化物歧化酶纯化条件的研究 　　摘要：目的研究重组人锰超氧化物歧化酶（rhMn－SOD）的纯化条件。方法超声法破碎细胞，用热变性方法沉淀蛋白质；优化DEAE离子交换层析的的分离条件，并用葡聚糖凝胶G100分子筛分离纯化蛋白质。结果菌体破碎最佳超声功率为300W，超声70个循环，粗酶液热处理最佳温度为75 ℃，时间10 min；DEAE上样缓冲液最佳pH值为9.0，洗脱液NaCl含量为0.1 mol/L ， G100纯化后，rhMn-SOD比活达到4 411.706 U/mg，纯化倍数是粗酶液的5倍。结论得到了适于分离纯化rhMn-SOD的较简便的工艺。 　　关键词：重组人锰超氧化物歧化酶；大肠杆菌；离子交换层析 　　中图分类号：Q554文献标识码：A文章编号：1672-979X(2007)07-0001-05 　　Study on Purification Condition of Recombinant Human Manganese Superoxide Dismutase 　　CHENG Yi1,CHEN Che-sheng2, WU Qian-yi2, ZHAO Jian1*,YUAN Qin-sheng1 　　(1. State Key Laboratory of Bioreactor Engineering, East China University of Science Technology, Shanghai 200237, China; 2. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China) 　　Abstract：Objective To study the purification method of recombinant human manganese superoxide dismutase(rhMn-SOD). Methods The cells were fragmented by ultrasonic. The protein was precipitated by the thermal denaturation. The condition of proteins separation by DEAE ion exchange chromatography was optimized, then Sephadex G-100 molecular sieving was used to purify protein. Results The optimization condition of ultrasonic power was 300 W, the cycles of ultrasonic were 70 cycles, the best thermal denaturation was 75 ℃and time was 10 minutes. The optimal pH value of loading buffer solution of DEAE was 9.0, the elution buffer was 0.1mol/LNaCl. After G-100, the specific activity of rhMn-SOD wa℃s 4 411.706 U/mg and it was 5-fold higher than crude enzyme extract. Conclusion The convenient method of separation and purification of rhMn-SOD is obtained. 　　Key words：recombinant human manganese superoxide dismutase; Escherichia coli; ion exchange chromatography 　　 　　超氧化物歧化酶（superoxide dismutase，SOD）是一类广泛存在于生物界的金属酶类[1]，是生物体防御活性氧（ROS）毒害的关键性防线，能催化超氧阴离子（O2.-）发生歧化反应，从而清除O2.-。具有抗衰老、防辐射及治疗炎症等一系列功能，已被广泛应用于食品及医疗保健行业[2]。真核细胞内的SOD主要有Cu，Zn，SOD和Mn-SOD 等。Mn-SOD存在于线粒体中，近年研究发现，Mn-SOD与炎症、衰老及肿瘤等多种疾病有关[3，4]。传统的Mn-SOD纯化方法较繁琐且费时费力，不利于规模化生产[5]。本课题组前期已经构建表达质粒