生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (2): 20-.doi: 10.3969/j.issn.2095-1736.2023.02.020

• 研究报告 • 上一篇    下一篇

人源SOD1及其突变体的表达纯化及酶学性质

杨方瑶， 简甜甜， 蒋立翔， 张　钰， 黄新河   

  1. 西南交通大学 生命科学与工程学院， 成都 610031
  • 出版日期:2023-04-18 发布日期:2023-04-18
  • 通讯作者: 黄新河，博士，副教授，硕士生导师，主要从事衰老与衰老相关疾病发生发展分子机制、抗衰老与衰老相关疾病药物学和新型抗衰品研发等方面的研究，E-mail: Xinhehuang@swjtu.edu.cn
  • 作者简介:杨方瑶，硕士，研究方向为基因工程，E-mail: yangfangy0@foxmail.com
  • 基金资助:
    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）

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

YANG Fangyao, JIAN Tiantian, JIANG Lixiang, ZHANG Yu, HUANG Xinhe   

  1. School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
  • Online:2023-04-18 Published:2023-04-18

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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800 μmol/L Cu2+和20 μmol/L Zn2+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。

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

Abstract: 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, 800 μmol/L Cu2+ and 20 μmol/L Zn2+ 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.

Key words: Cu/Zn superoxide dismutase, Escherichia coli, site-directed mutagenesis, recombinant expression and purification, activity

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