Development of Stable Superoxide Dismutase by Protein Engineering

超氧化物歧化酶(SOD)通过减少体内产生的超氧阴离子自由基的浓度来保护生物免受氧化损伤。因此，临床上考虑对有炎症和缺血情况的患者给予全身和局部给药。然而，尽管超氧化物歧化酶对热和变性剂相对稳定，但它很容易被酶反应的产物过氧化氢灭活，因此我们试图通过克隆人铜锌超氧化物歧化酶的基因并通过基因工程技术将氨基酸替换引入到超氧化物歧化酶中来获得稳定的人铜锌超氧化物歧化酶。从人胎盘cDNA文库中分离得到铜锌超氧化物歧化酶克隆pSOD2，构建了含有结肠素E1启动子的表达载体pUBE2，并将其导入大肠杆菌W3110。人重组超氧化物歧化酶经丝裂霉素C处理后，占大肠杆菌总蛋白的12%，以pUBE2、pUBE118为模板，构建了可用于定点突变的表达载体pUBE118，并与人工合成的寡核苷酸在Arg-143上构建了分别取代His、Asn和Asp的突变型SOD表达载体。将这些质粒分别导入大肠杆菌TG1并用丝裂霉素C处理细胞后，所有突变的超氧化物歧化酶都在大肠杆菌细胞中产生了与野生型酶相似的量。从电泳后的活性染色结果来看，带有His替换的SOD的活性约为野生型的1/10，而带有Asn替换的蛋白质的活性仅为野生型的1/100，而Asp替换的蛋白质中没有检测到活性。因此，取代其Arg-143的超氧化物歧化酶有可能成为稳定的超氧化物歧化酶的候选者。

Superoxide dismutase (SOD) protects a living body against oxidative damages by reducing the concentration of superoxide radical generated in the body. Therefore, general and local administrations of SOD are considered clinically to patients with inflamations and ischemic conditions. However, SOD is easily inactivated by hydrogen peroxide, a product of the enzyme reaction, although it is relatively stable against heat or denaturating agents.We intended to develop a stable human Cu, Zn-SOD by cloning its cDNA and introducing amino acid replacements into SOD through gene engineering techniques. From pSOD2, a Cu, Zn-SOD clone which was isolated from a human placenta cDNA library, we constructed an expression plasmid, pUBE2 which uses the colicin E1 promoter and introduced it into E. coli W3110. After mitomycin C treatmentment, the human recombinant SOD amounted to 12% of the total E. Coli proteins.From pUBE2, pUBE118, a plasmid for the use of site-directed mutagenesis was made, and with this plasmid and synthetic oligodeoxynucleotides, a series of expression plasmid which direct mutated SODs having replacements of His, Asn and Asp, respectively, at Arg-143. After introducing each of these plasmids into E. Coli TG1 and treating the cells with mitomycin C, all of the mutated SODs were poduced in E. Coli cells to amounts similar to the wild-type enzyme. From the results of activity staining after electrophoresis, SOD with His replacement had about one tenth of the activity of the wild type, while the protein with Asn replacement showed one hundredth activity and no activity was detected in the Asp replacement. Thus the SOD with his replacent at Arg-143 will be Valuable as a candidate of the stable SOD.