X-ray Crystallographic Study of Antibiotics resistant Proteins
| Project/Area Number |
17590090
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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 |
Drug development chemistry
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| Research Institution | Josai International University |
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Principal Investigator |
NUKAGA Michiyoshi Josai International University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 教授 (20251150)
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| Co-Investigator(Kenkyū-buntansha) |
KAKEGAWA Tomohito Josai International University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (80169391)
OHUCHI Nozomi Josai International University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (20398556)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | Infection Disease / Antibiotics / Enzyme / Protein / X-ray Crystallography |
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| Research Abstract |
4 Projects are included in this study.
1) β-Lactamase:
1-a) SHV-1 : meropenem complex:
High resolution (1.05 A) x-ray structure of SHV-1 covalently complexed with meropenem was solved. High resolution data provides some electron densities for important hydrogen atoms. One of the interesting results is protonated Glu166 and associated hydrogen bonding network around deacylation water. They suggest the deacylation water with the nucleophilicity decreased.
1-b) GC1 β-Lactamase : ampicillin complex:
Complex of class C β-lactamase from Enterobacter cloacae GC1 with good substrate ampicillin was trapped using protein modification and solved by x-ray crystallography. This is the first report in which acyl-enzyme of substrate could be trapped in classC β-lactamase.
2) AAC/APH bifunctional enzyme:
AAC/APH bifunctional enzyme was from MRSA and it add acetyl or/and phosphate group to amino glycoside antibiotics to inactivate. AAC/APH bifunctional enzyme gene was cloned to pCold vector for high expression. 3-5 mg protein could be obtained from 1 L culture. Kanamycin affinity column and gel filtration were used for purification. Preliminary crystallization screening was done. Although some crystals could be obtained, further optimizations are required.
3) Macrolide phosphotransferase:
Macrolide phosphotransferase gene (mphA) was cloned using PCR to pCold vector for high expression. Currently, the amount of the enzyme is not enough to make crystals. On the other hand, regulatory protein, mphR, could be highly expressed as GST fusion protein.
4) Dihydropteroate synthase from Mycobacterium Jeprae:
Dihydropteroate synthase (DHPS) is main target of dapson which is the important anti-M. leprae drug. To solve the complex of DHPS-dapson, high expression systems in E.coli were tested. In all of case, DHPS was expressed as inclusion body at first, co-expression with chaperone protein was effective to reduce them.
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Report
(3 results)
Research Products
(15 results)
