| Project/Area Number |
13557161
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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 |
Conservative dentistry
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
FUKUI Kazuhiro Okayama University, Graduate School of Medicine and Dentistry, Professor, 大学院・医歯学総合研究科, 教授 (70034171)
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| Co-Investigator(Kenkyū-buntansha) |
INOUE Tetsuyoshi Okayama University, Graduate School of Medicine and Dentistry, Research associate, 大学院・医歯学総合研究科, 助手 (20223258)
MAEDA Hiroshi Okayama University, Graduate School of Medicine and Dentistry, Research associate, 大学院・医歯学総合研究科, 助手 (00274001)
KOKEGUCHI Susumu Okayama University, Graduate School of Medicine and Dentistry, Assistant Professor, 大学院・医歯学総合研究科, 助教授 (10144776)
NISHIMURA Fusanori Okayama University, Graduate School of Medicine and Dentistry, Assistant Professor, 大学院・医歯学総合研究科, 助教授 (80208222)
KODAMA Takao Kyusyu Institute of Technology, Bioinformatics, Professor, 情報工学部, 教授 (30034200)
小西 法文 岡山大学, 歯学部附属病院, 講師 (60243466)
村山 洋二 岡山大学, 大学院・医歯学総合研究科, 教授 (50029972)
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| Project Period (FY) |
2001 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2001: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | insoluble glucan synthease / oral streptococci / dextran / secondary structure / GTF-I / Streptococcus / catalytic domain / crystallization / グルカン合成酵素 / 口腔レンサ球菌 / 立体構造解析 / GTF / X線結晶解析 / 大量発現系 / 立体構造 / 不溶性グルカン合成酵素 |
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| Research Abstract |
Insoluble glucan synthetase (GTF-I) of Streptococcus sobrinus, a member of oral streptococci, is an enzyme that splits sucrose into glucose and fructose and synthesizes water-insoluble glucan. For structure-function studies of this enzyme, we constructed overexpression systems of the GTF-I and its deletion mutants, established efficient purification methods, and investigated optimum conditions for crystallization. The GTF-I is composed two domains, N-terminal catalytic domain (GS) and C-terminal dextran-binding domain (DXB). Prior to crystallization, we constructed the Escherichia coli expression systems of three polypeptides, the complete peptide GTF-I, the N-terminal 83 amino acids-deleted GS-6R, and the only catalytic domain GS. Each polypeptide was highly purified with high efficiencies by combinations of ammonium sulfate precipitation, ion-exchange chromatography, affinity chromatography, and gel-filtration chromatography. Optimum conditions (pH, salt concentrations and the presence of precipitant agents) for crystallization were screened for each polypeptide using hanging-drop vapor diffusion method. In the complete GTF-I, micro-crystals were obtained in a pH range of pH 6-8 and in salt concentrations of 0-0.2 M. However, crystal formation of GS-6R protein was not observed in any conditions tested. In GS, many micro-crystals were formed in similar conditions to those in GTF-I. Furthermore, to reduce the effects of the flexibility of DXB domain, crystallization was performed at low temperature or at the presence of dextran. However, the quality of crystals was not improved. Although crystals suitable for X-ray structural analysis have not yet been obtained, further screening will lead to achieve high-quality crystals of this enzyme.
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