Large production and purification of glutamate decarboxylase and application for diagnosing diabetes mellitus
| Project/Area Number |
10556018
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
UENO Hiroshi Kyoto University, Agriculture, Associate Professor, 農学研究科, 助教授 (30241160)
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| Co-Investigator(Kenkyū-buntansha) |
TAKEICHI Yoichiro Taiho Pharmaceutical Co. Pharmacokinetic Research Lab., Director, 製剤研究所, 所長(研究職)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥8,300,000 (Direct Cost: ¥8,300,000)
Fiscal Year 1999: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1998: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | decarboxylase / GABA / diabetes / diagnosis |
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| Research Abstract |
Expression system of an isozyme of rat brain glutamate decarboxylase, GAD65, was constructed with Saccharomyces cerevisiae. GAD65 is found to be a target protein for autoantibody produced in type I diabetic patients. This autoantibody exists in the early stages of diabetic development, as early as 10 year prior to showing the diabetic symptom. Therefore, our aim was to obtain a large amount of GAD65 which might be used in the diagnosis of the diabetes.
We have optimized the growth conditions by varying the carbon source, replacing the promoter and terminator sequences, and altering the inducing materials. We have examined thermal stability of the expressed protein, in which we found that competitive inhibitors significantly protect the protein from the thermal inactivation. Providing a stable enzyme is one of the requirements for the development of diagnostic agents. We have also developed an apparatus for convenient assaying the enzymatic activity in minute quantity. Combination of micro-capillary tube and photo-operated counter gave promising results. We are currently working to optimize the apparatus.
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Report
(3 results)
Research Products
(6 results)
