Pan Jianru, Chen Lijuan, He Huocong, Su Ying, Wang Xiangling, Li Xian, Chen Cuihuang, Wu Lunqiao, Liu Shutao
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, Fujian, China.
Laboratory of Radiation Oncology and Radiobiology, Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, Fujian Key Laboratory of Tumor Translational Cancer Medicine, Fuzhou 350014, Fujian, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jul 25;33(7):1168-1177. doi: 10.13345/j.cjb.170007.
Superoxide dismutase (SOD) family is necessary to protect cells from the toxicity of reactive oxygen species produced during normal metabolism. Among SODs, manganese-containing superoxide dismutase (Mn-SOD, SOD2) is the most important one. The DNA fragment containing the full nucleotide of full-length human SOD2 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with tag GST. DNA construct was then transformed into Escherichia coli BL21 (DE3) and expression was induced with IPTG at 25 ℃. The recombinant fusion protein GST-SOD2 (46 kDa) was purified from the bacterial lysate by GST resin column affinity chromatography. GST tag was cleaved with thrombin, and a crude SOD2 recombinant protein (25 kDa) was obtained and further purified by heparin affinity chromatography. Activities of the two SOD2 proteins were 1 788 and 2 000 U/mg, respectively. Both SOD2 proteins were stable under physiological condition and cell-penetrating (P<0.05). Our findings open the possibility to study the structure and effects of two full-length recombinant SOD2 proteins.
超氧化物歧化酶（SOD）家族对于保护细胞免受正常代谢过程中产生的活性氧物种的毒性至关重要。在超氧化物歧化酶中，含锰超氧化物歧化酶（Mn-SOD，SOD2）是最重要的一种。合成了包含全长人SOD2完整核苷酸的DNA片段，并将其插入带有标签GST的原核表达载体pGEX-4T-1中。然后将DNA构建体转化到大肠杆菌BL21（DE3）中，并在25℃用IPTG诱导表达。通过GST树脂柱亲和层析从细菌裂解物中纯化重组融合蛋白GST-SOD2（46 kDa）。用凝血酶切割GST标签，获得粗制的SOD2重组蛋白（25 kDa），并通过肝素亲和层析进一步纯化。两种SOD2蛋白的活性分别为1788和2000 U/mg。两种SOD2蛋白在生理条件下均稳定且具有细胞穿透性（P<0.05）。我们的研究结果为研究两种全长重组SOD2蛋白的结构和作用提供了可能性。
