| Project/Area Number |
13671261
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | St. Marianna University School of Medicine |
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Principal Investigator |
FUKUDA Mamoru St. Marianna University School of Medicine, Department of Surgery, Professor, 医学部, 助教授 (50081724)
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| Co-Investigator(Kenkyū-buntansha) |
OHTA Tomohiko St. Marianna University School of Medicine, Department of Surgery, Assistant Professor, 医学部, 講師 (60233136)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | gene therapy / protein degradation / scFv / ubiquitin ligase / p53 / RING finger / scFv |
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| Research Abstract |
We have attempted to create an engineered ubiquitin ligase with ScFv as a substrate recognition site that target and degrade only p53 mutant but not the wild type. The ScFv was cloned from cDNA library of a hybridoma cells that express Pab240, an antibody recognizes mutant p53 such as R273P, R175H, or V143A. We first tested a double RING finger ubiquitin ligase whether it actually targets the intended specific substrates. The engineered ligase contains the RING finger domains of both BRCA1 and BARD1 linked to a substrate recognition site PCNA, which is known to interact with cyclin dependent kinase inhibitor p57. The double RING finger ubiquitin ligase formed a homo-oligomer complex and exhibited significant ligase activity. Co-transfection of the ligase reduced the expression of transfected p57 to the back ground level in proteasome dependent manner, and restored the colony formation ability of U2OS cells that is otherwise inhibited by overexpressed p57. The results indicate the ability of the engineered double RING ubiquitin ligase to target the intended substrate. Next we tested the double RING ubiquitin ligase linked to the ScFv cloned from Pab240. However so far it has not been successful most likely because of the affinity problem between the ScFv and p53 mutants. To overcome the problem a new ScFv has been made by DNA synthesize method.
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