Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, 86 Asanbyeongwon-gil, Seoul 138-736, South Korea.
Appl Biochem Biotechnol. 2013 Mar;169(5):1633-47. doi: 10.1007/s12010-012-0025-x. Epub 2013 Jan 19.
Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction of EC-SOD is linked to many diseases, suggesting that the protein may have therapeutic value. EC-SOD is reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichia coli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding during purification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human EC-SOD (hEC-SOD) was purified by MBP affinity and anion exchange chromatography, and its identity was confirmed by MALDI-TOF MS analysis. The purified protein showed good enzyme activity in vitro; however, there was a difference in metal binding. When copper and zinc were incorporated into hEC-SOD before MBP tag cleavage, the enzymatic activity was higher than when the metal ions were bound to the purified protein after MBP tag cleavage. Therefore, the enzymatic activity of hEC-SOD is associated with metal incorporation and protein folding via disulfide bond.
细胞外超氧化物歧化酶（EC-SOD）是唯一能在细胞外空间清除超氧自由基的酶。EC-SOD 的还原与许多疾病有关，这表明该蛋白可能具有治疗价值。据报道，EC-SOD 在大肠杆菌细胞质中过表达时不溶，并形成包含体。复性过程的优点是产量高，但在纯化过程中经常会发生聚集或错误折叠。本研究首次表明，与麦芽糖结合蛋白（MBP）、氮源利用物蛋白 A 和蛋白二硫键异构酶融合，可使 EC-SOD 在大肠杆菌细胞质中可溶性过表达。MBP 标记的人 EC-SOD（hEC-SOD）通过 MBP 亲和和阴离子交换层析进行纯化，并通过 MALDI-TOF MS 分析确认其身份。纯化的蛋白质在体外表现出良好的酶活性；然而，金属结合存在差异。当铜和锌在 MBP 标签切割前结合到 hEC-SOD 中时，酶活性高于 MBP 标签切割后结合到纯化蛋白上的金属离子。因此，hEC-SOD 的酶活性与金属结合和通过二硫键折叠有关。
