重组人SOD23 杂合酶原核表达及纯化方法的研究.pdf

食品与药品 Food and Drug 2011年第 13卷第 11期 385 重组人SOD2/3杂合酶原核表达及纯化方法的研究 * 周大海，范立强 ，赵 健，程安阳 （华东理工大学生物反应器工程国家重点实验室，上海 200237 ） 摘 要：目的 摸索工程菌的表达条件并分离纯化，获得高活性的重组人SOD2/3杂合酶 （rhSOD2/3 ）。方法 利用含有质粒 pET-28a-SOD2/3 的工程菌诱导表达rhSOD2/3，考察诱导温度、诱导剂浓度、Mn2+浓度与加入时机对重组酶表达量与活性 的影响；利用金属螯合亲和层析纯化rhSOD2/3 。结果 重组酶的表达量占菌体总蛋白40 %~50 %，最佳表达温度15 ℃、诱 导剂浓度0.5 mmol/L, Mn2+浓度2.5 mmol/L ，表达时间16 h ，亲和层析能较好的纯化rhSOD2/3，比活从粗酶液220 U/mgpr提 高到930 U/mgpr 以上。结论 成功获得了rh SOD2/3最适的表达条件及纯化方法。 关键词：SOD2/3杂合酶；表达；纯化；亲和层析 中图分类号：Q538 文献标识码：A 文章编号：1672-979X(2011)11-0385-04 Study on Expression and Purification of Recombinant Human SOD2/3 Heterozyme * ZHOU Da-hai, FAN Li-qiang , ZHAO Jian,CHENG An-yang (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China) Abstract: Objective To obtain high expression of recombinant human SOD2/3 heterozyme (rhSOD2/3) by optimizing the expression condition . Methods The rhSOD2/3 heterozyme was expressed by the recombinant engineering bacteria containing pET-28a-SOD2/3 plasmid. The effects of induction temperature, concentration of inductor and Mn2+ , adding time were investigated on the expression amount and activity of rhSOD2/3 heterozyme. Then, the rhSOD2/3 heterozyme was purified using metal chelate affinity chromatography. Results The rhSOD2/3 heterozyme was expressed successfully, accounting for 40 %~50 % of the total proteins. The optimal expression condition was the expression induced by 0.5mmol/L IPTG and 2.5mmol/L Mn2+ at 15℃ for 16h. The specific activity of rhSOD2/3 was 220 U/mgpr in crude enzymes, while over 930 U/mgpr after purified by affinity chromatography.