Development of Stable Superoxide Dismutase by Protein Engineering

超氧化物歧化酶（SOD）通过降低体内产生的超氧化物自由基的浓度来保护生物体免受氧化损伤。因此，临床考虑对炎症和缺血性疾病患者进行全身和局部SOD管理。然而，SOD很容易被过氧化氢灭活，过氧化氢是酶反应的产物，尽管它对热或变性剂相对稳定。本研究拟通过克隆铜、锌超氧化物歧化酶cDNA，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，构建稳定的人铜、锌超氧化物歧化酶。以从人胎盘cDNA文库中分离到的Cu， Zn-SOD克隆pSOD2为载体，构建了含有colicin E1启动子的表达质粒pUBE2，并将其导入大肠杆菌W3110。丝裂霉素C处理后，人重组SOD占大肠杆菌总蛋白的12%。从pUBE2、pUBE118制备了用于位点定向诱变的质粒，并用该质粒和合成的寡脱氧核苷酸组成了一系列表达质粒，这些表达质粒在Arg-143位点分别替换了His、Asn和Asp。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，大肠杆菌细胞中产生了与野生型酶相似的所有突变sod。从电泳后的活性染色结果来看，His取代的SOD活性约为野生型的十分之一，Asn取代的蛋白活性为野生型的百分之一，Asp取代的蛋白未检测到活性。因此，在Arg-143位点有其替代物的超氧化物歧化酶作为稳定超氧化物歧化酶的候选物是有价值的。

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.