Eiamphungporn Warawan, Yainoy Sakda, Prachayasittikul Virapong
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Iran J Biotechnol. 2016 Dec;14(4):243-249. doi: 10.15171/ijb.1465.
Human Cu/Zn superoxide dismutase (hSOD1) is an antioxidant enzyme with potential as a therapeutic agent. However, heterologous expression of hSOD1 has remained an issue due to Cu insufficiency at protein active site, leading to low solubility and enzymatic activity.
The effect of co-expressed human copper chaperone (hCCS) to enhance the solubility and enzymatic activity of hSOD1 in was investigated in the presence and absence of Cu.
and were constructed and individually transformed into strain BL21(DE3). The recombinant hSOD1 was expressed and purified using immobilized metal affinity chromatography. The yield and specific activity of hSOD1 in all conditions were studied.
Co-expression with hCCS increased hSOD1 solubility at 37°C, but this effect was not observed at 25°C. Notably, the specific activity of hSOD1 was enhanced by 1.5 fold and greater than 3 fold when co-expressed with hCCS at 25°C with and without Cu supplement, respectively. However, the chaperone co-expression did not significantly increase the yield of hSOD1 comparable to the expression of hSOD1 alone.
This study is the first report demonstrating a potential use of hCCS for heterologous production of hSOD1 with high enzymatic activity.
人铜锌超氧化物歧化酶（hSOD1）是一种具有作为治疗剂潜力的抗氧化酶。然而，由于蛋白质活性位点的铜不足，hSOD1的异源表达一直是个问题，导致其溶解度和酶活性较低。
研究在有铜和无铜的情况下，共表达人铜伴侣蛋白（hCCS）对提高hSOD1在大肠杆菌中的溶解度和酶活性的影响。
构建质粒并分别转化到大肠杆菌BL21(DE3)菌株中。使用固定化金属亲和色谱法表达并纯化重组hSOD1。研究了所有条件下hSOD1的产量和比活性。
与hCCS共表达可提高hSOD1在37°C时的溶解度，但在25°C时未观察到这种效果。值得注意的是，在25°C有铜和无铜补充的情况下，与hCCS共表达时，hSOD1的比活性分别提高了1.5倍和超过3倍。然而，与单独表达hSOD1相比，伴侣蛋白共表达并没有显著提高hSOD1的产量。
本研究首次报道了hCCS在异源生产具有高酶活性的hSOD1方面的潜在用途。
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