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) 家族是保护细胞免受正常代谢过程中产生的活性氧 (ROS) 毒性所必需的，含Mn²⁺离子的超氧化物歧化酶 (Mn-SOD，SOD2) 是其中最重要的一种。本研究合成了人源SOD2 全基因序列，并将其插入带有GST 的原核表达载体pGEX-4T-1 中，成功构建了GST-SOD2 融合蛋白表达质粒。然后，将重组质粒pGEX-4T-1-SOD2 转化大肠杆菌BL21 (DE3)，用IPTG 在25 ℃下诱导表达融合蛋白，得到可溶性GST-SOD2 融合蛋白，经GST 亲和树脂纯化得到比活为1 788 U/mg 的纯蛋白，分子量约为46 kDa。利用凝血酶切去GST 标签后经肝素亲和柱纯化得到了电泳纯的SOD2 重组蛋白，该蛋白分子量约为25 kDa，与SOD2全长序列的理论分子量相符，比活为2 000 U/mg。两种重组SOD2 蛋白在生理条件下都具有良好的SOD 活性，且都具有显著的跨膜能力 (P<0.05)。这些工作为深入研究两种全长重组SOD2 蛋白的结构与生物效应建立了基础。.
Keywords: Mn-SOD; construction; expression and purification; fusion protein; stability; transduction efficiency.