重组超氧化物歧化酶13杂合酶在大肠杆菌中表达优化.docVIP

重组超氧化物歧化酶13杂合酶在大肠杆菌中表达优化 　　摘要：目的获取活性高的重组SOD 1/3杂合酶。方法用含pET-SOD 1/3的工程菌诱导表达重组SOD 1/3杂合酶，通过分析SDS及SOD酶活性，考察诱导温度、诱导时间、诱导剂浓度、Cu2+、Zn2+浓度对重组酶表达量和活性的影响。结果正交试验表明，用1 mmol/L 异丙基-β-D-硫代半乳糖苷（IPTG）在A600 =1.2时诱导，Cu2+、Zn2+浓度分别为2.4 mmol/L，诱导温度15 ℃，诱导时间12 h，能获得高活性重组SOD 1/3，其比活性为1 681 U/mg。结论培养温度和Cu2+、Zn2+浓度是优化表达的最主要因素。降低诱导温度有利于重组酶的可溶性表达，添加Cu2+、Zn2+能促进重组酶活性提高。 　　关键词：超氧化物歧化酶；杂合酶；表达；优化 　　中图分类号：Q554文献标识码：A文章编号：1672-979X(2008)11-0008-04 　　 　　Optimal Expression of Recombinant Hybrid Enzyme SOD1/3 in E. coli 　　SONG Xiao-ling, FAN Li-qiang*, RONG Zhi-feng, ZHAO Jian*, LI Su-xia, YUAN Qin-sheng 　　(State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China) 　　Abstract：Objective To obtain recombinant hybrid enzyme SOD1/3 with higher activity in E. coli. Methods The recombinant bacteria containing expression plasmid pET-SOD1/3 was used to express hybrid enzyme SOD1/3. The effects of induction temperature, induction time, IPTG concentration and Cu2+ or Zn2+ concentration on the expression amount and activity of recombinant hybrid enzyme SOD1/3 were investigated by SDSand SOD activity assay. Results When the recombinant bacteria were cultured at 15℃ for 12 h with 2.4 mmol/L Cu2+ or Zn2+ and 1 mmol/L IPTG at A600=1.2, the activity of hybrid enzyme SOD1/3 was higher with specific activity of 1 681U/mg. Conclusion The optimal expression of hybrid enzyme SOD1/3 is mainly affected by induction temperature and Cu2+ or Zn2+ concentration. Hybrid enzyme SOD1/3 can be expressed in supernatant at low temperature. The addition of Cu2+ or Zn2+ can increase the activity of hybrid enzyme SOD1/3. 　　Key words：superoxide dismutase; hybrid enzyme; expression; optimization 　　 　　超氧化物歧化酶（superoxide dismutase，SOD）是催化超氧阴离子（O2?-）歧化反应的金属酶。人体内SOD有3种：细胞内Cu, ZnSOD（SOD1）、线粒体中MnSOD（SOD2）和胞外Cu, Zn EC-SOD（SOD3）。作为O2?-清除剂，SOD广泛用于防辐射、抗衰老、防治肿瘤等方面[1,2]。目前，对Cu, ZnSOD的研究较深入全面，但其体内半衰期短（6min），不能与内皮细胞特异性的结合等，限制了它的应用。EC-SOD恰能弥补这些不足，但它在原核和酵母系统中的表达效果不理想[3-5]，不能大量制备。为此