Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-06-18 DOI:10.1016/j.pep.2024.106535

Chengli Zhao , Wenrui Huang , Jiayi Su , Xinshuang Zhang , Jingli Xue , Cailiang Zhang , Juan Han , Yang Zhou , Yun Wang

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引用次数: 0

Abstract

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH₄)₂SO₄ was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

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重组人超氧化物歧化酶与resilin-like多肽整合的纯化与特征。

人体超氧化物歧化酶（hSOD1）在体内有氧代谢和消除自由基的过程中发挥着重要作用。然而，现有 SOD 的生产面临着纯化方法复杂、成本高、产品稳定性差等问题。本实验通过硫酸铵沉淀法实现了 hSOD1 的低成本、快速和简单纯化，并实现了重组蛋白的耐热性。我们构建并表达了含有resilin-like多肽标签的重组蛋白hSOD1-LR。结果表明，1.5 M (NH4)2SO4的纯化效果最好，纯化后酶活性恢复率达80%。然后，根据其热稳定性，在 60 ℃条件下进一步纯化该兴趣蛋白，发现纯化倍数可达 24 倍，纯化效果与镍柱亲和层析纯化的 hSOD1-6xHis 相似。hSOD1-LR 的稳定性表明，重组蛋白 hSOD1-LR 比 hSOD-6xHis 具有更好的稳定性。同时，hSOD1-LR 在 pH 值为 5.5 时的活性接近 80%。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.