1988 Fiscal Year Final Research Report Summary
Development of Stable Superoxide Dismutase by Protein Engineering
| Project/Area Number |
62870015
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Yamaguchi University School of Medicine |
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Principal Investigator |
NAKAZAWA Atsushi Yamaguchi University School of Medicine (Professor), 医学部, 教授 (90025594)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAHARA Hiromi Ube Industries,Ltd., Ube Institute (Principal Investigator), 宇部研究所, 主任研究員
YAMADA Mamoru Yamaguchi University School of Medicne (Research Associate), 医学部, 助手 (30174741)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Superoxide dismutase / cDNA / Colicin E1 promoter / プロモーター / 部位特異的変異法 |
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| Research Abstract |
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.
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Research Products
(8 results)
