重组人SOD2/3杂合酶原核表达及纯化方法的研究

Study on Expression and Purification of Recombinant Human SOD2/3 Heterozyme

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。

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℃ for16h.The specific activity of rhSOD2/3 was 220 U/mgpr in crude enzymes,while over 930 U/mgpr after purified by affinity chromatography.Conclusion This study can show the optimized expression condition and purification method of rhSOD2/3 heterozyme.