人源SOD1及其突变体的表达纯化及酶学性质  

Expression,purification and characterization of human superoxide dismutase 1 and itsmutants

作　　者：杨方瑶 简甜甜 蒋立翔 张钰[1] 黄新河[1] YANG Fangyao;JIAN Tiantian;JIANG Lixiang;ZHANG Yu;HUANG Xinhe(School of Life Science and Engineering,Southwest Jiaotong University,Chengdu 610031,China)

机构地区：[1]西南交通大学生命科学与工程学院,成都610031

出　　处：《生物学杂志》2023年第2期20-26,共7页Journal of Biology

基　　金：中央高校基本科研业务费项目(No.2682020ZT97);大学生科研创新训练计划项目(No.202010613061)。

摘　　要：分析人源SOD1(hSOD1)、牦牛SOD1序列,定点突变得到hSOD1突变体Mt1SOD1(E25G、P29T、E101V、C112S)和Mt2SOD1(I18T、N20H、K31V、C112S),全合成经密码子优化的hSOD1、Mt1SOD1、Mt2SOD1、牦牛SOD1序列后,将其重组克隆到表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3)获得重组菌。重组菌经1 mmol/L IPTG诱导,在含800μmol/L Cu^(2+)和20μmol/L Zn^(2+)的LB培养基中,25℃、180 r/min诱导培养16 h,4种SOD1均以可溶性形式高效表达,镍亲和层析可有效纯化重组SOD1。100 mL LB摇瓶培养获得的hSOD1活性为71094 U/mg,产率为4.57 mg。Mt1SOD1活性为128506 U/mg,产率为3.13 mg。Mt2SOD1活性为58700.1 U/mg,产率为5.47 mg。牦牛SOD1活性为42969.5 U/mg,产率为6.81 mg。对hSOD1、Mt1SOD1的酶学性质研究表明,在25℃~55℃,酶活力基本保持不变,75℃保温30 min,hSOD1相对酶活力保持在50%左右,Mt1SOD1保持30%酶活性。在pH 3.6~10.4条件下放置30 min,两种SOD1均能保持70%以上的酶活力。研究通过定点突变获得高活性高稳定性的hSOD1和Mt1SOD1,为SOD1在医疗、保健、化妆品等领域的应用奠定基础。SOD is an important functional protein and industrial enzyme,and its source is a hot topic in the field.Here hSOD1 and bosgrunniens SOD1 were analyzed by bioinformatics firstly,and Mt1SOD1(E25G,P29T,E101V,C112S)and Mt2SOD1(I18T,N20H,K31V,C112S)were obtained by site-directed mutagenesis.Condons-optimized hSOD1,Mt1SOD1,Mt2SOD1,and bosgrunniens SOD1 were totally synthesized and then cloned into pET-28a(+)to achieve recombinant plasmid for transforming into E.coli BL21(DE3).The four kinds of SOD1 were induced and expressed successfully under the conditions of 1 mmol/LIPTG,800μmol/L Cu^(2+)and 20μmol/L Zn^(2+)in LB medium at 25℃and 180 r/min for 16 h.Next,Ni-NTA was used to purify four recombinant SOD1s.The activity of hSOD1 obtained from 100 mL LB was 71094 U/mg,and the yield was 4.57 mg.The activity of Mt1SOD1 was 128506 U/mg,and the yield was 3.13 mg.The activity of Mt2SOD1 was 58700.1 U/mg,and the yield was 5.47 mg.The activity of bos grunniens SOD1 was 42969.5 U/mg,and the yield was 6.81 mg.The enzymatic activity of hSOD1 and Mt1SOD1 remained no change between 25℃and 55℃,and the relative enzymatic activity of hSOD1 remained about 50%after incubation at 75℃for 30 minutes,and that of Mt1SOD1 remained 30%.Under the condition of pH 3.6-10.4 for 30 min,both SOD1s can maintain more than 70%of the enzyme activity.hSOD1 and Mt1SOD1 with high activity and high stability were obtained by site-directed mutagenesis,which laid the foundation for the application of SOD1 in medical,health care,cosmetics and other fields.

关 键 词：铜锌超氧化物歧化酶 大肠杆菌 定点突变 重组表达与纯化 活性 

分 类 号：Q78[生物学—分子生物学] Q55