As the most important member of antioxidant defense system, human Cu,Zn superoxide dismutase (hCu,Zn-SOD) protects cells against the free radicals produced by aerobic metabolism. hCu,Zn-SOD has been widely used in food, cosmetic and medicine industry due to its health benefits and therapeutic potentials. However, a more extensive application of hCu,Zn-SOD is limited by the challenge of expensive and low production of high-activity hCu,Zn-SOD in large scale. In this study, the codon-optimized hCu,Zn-SOD gene was synthesized, cloned into pET-28a( +) and transformed into Escherichia coli BL21(DE3). After induction with IPTG or lactose, hCu,Zn-SOD was highly expressed as soluble form in LB medium with 800 μM Cu2+ and 20 μM Zn2+ at 25 °C. The recombinant hCu,Zn-SOD was efficiently purified by nickel affinity chromatography. Through optimization of fed-batch fermentation conditions, 342 mg purified hCu,Zn-SOD was obtained from 1 L cultures fermented in a 3-L bioreactor. Furthermore, the recombinant hCu,Zn-SOD retained the enzymatic specific activity of 46,541 U/mg. This study has opened up an effective avenue for industrial production of hCu,Zn-SOD through microbial fermentation in the future.

中文翻译：

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高活性人铜锌超氧化物歧化酶在大肠杆菌中的高水平可溶性表达

作为抗氧化防御系统最重要的成员，人类铜、锌超氧化物歧化酶（hCu,Zn-SOD）保护细胞免受有氧代谢产生的自由基的侵害。hCu,Zn-SOD 由于其对健康的益处和治疗潜力，已广泛应用于食品、化妆品和医药行业。然而，hCu,Zn-SOD 的更广泛应用受到高活性 hCu,Zn-SOD 大规模生产昂贵和低产量的挑战。本研究合成了密码子优化的 hCu,Zn-SOD 基因，克隆到 pET-28a(+) 并转化大肠杆菌 BL21(DE3)。用 IPTG 或乳糖诱导后，hCu、Zn-SOD 在 25 °C 下在含有 800 μM C​​u2+ 和 20 μM Zn2+ 的 LB 培养基中以可溶形式高度表达。重组 hCu,Zn-SOD 经镍亲和层析有效纯化。通过优化补料分批发酵条件，在 3-L 生物反应器中发酵的 1 L 培养物获得了 342 mg 纯化的 hCu,Zn-SOD。此外，重组 hCu,Zn-SOD 保留了 46,541 U/mg 的酶比活性。该研究为今后微生物发酵工业化生产hCu、Zn-SOD开辟了一条有效途径。