| Project/Area Number |
22590270
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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 medical chemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
ASADA Hidetsugu 京都大学, 大学院・医学研究科, 特定助教 (20399041)
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| Co-Investigator(Kenkyū-buntansha) |
SHIROISHI Mitsunori 九州大学, 大学院・薬学研究科, 助教 (00380527)
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| Project Period (FY) |
2010 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2012: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2011: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | 構造生物 / GPCR / 抗体 / 結晶化 / X線結晶構造解析 / 共結晶 / 構造解析 |
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| Research Abstract |
Angiotensin II receptor type2 (AT2) play an importantrole for regulating blood pressure. Recent studies strongly suggest that AT2 not only mediates blood pressure control but also plays an important role in cardiovascular protection such as anti cardiac hypertrophy. To solve AT2 structure is important for understanding of the molecular mechanism of blood pressure control and developing of drug against cardiac hypertrophy. Recently, several GPCR structures have been solved. However, to determine of GPCR crystal structure still remains having some problems. In this study, we developed AT2 structure specific recognition antibodies to overcome these problems. We have been trying to crystallize of AT2 with Fab in order to solve the structure in this study. Stabilized AT2 mutant (AT2-T4L) has been constructed by deletion of the flexible N- and C-terminus and insertion of T4 lysozyme (T4L) to the third intracellular loop (i3). AT2-T4L has been expressed in Sf9 insect cells. The expression lev
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els of AT2-T4L have been assessed by flow cytometric analysis (FACS) and specific ligand-binding assay. We have purified from the cell membrane expressing AT2-T4L using TALON resin and size exclusion chromatography (SEC). The characteristics of the purified receptors have been assessed by thermal stability (CPM) assay. The antibodies have been developed from immunization of mice with wild type AT2. The antibodies have been screened using liposome-ELISA, dot plot analysis, FACS, and fluorescence size exclusion chromatography (FSEC). We have prepared Fab region from the screened antibodies for crystallization. The purified AT2T4L has been incubated with its Fab. Then, the complex has been purified by means of SEC. The complex has been crystallized in vapordiffusion. The AT2-T4L could be expressed and purified successfully. The expression level was 57.3±8.9% (Average±SD) of whole infected cells (FACS) and 91.7±37.8pmol/mg -membrane (ligand-binding assay). The thermal stability of AT2-T4L had melting temperature (Tm) of 34 and 58℃. This data show that thermal stability of AT2-T4L is not enough for crystallization. Actually, we could not acquire the crystals from this sample in LCP. To contribute improvement of AT2-T4L stability, we have established three AT2 specific antibodies via the screening steps. The complex of AT2-T4L and Fab had Tm of 62℃. This thermal stability was higher than AT2-T4L. Furthermore, the thermal stability of complex was maintained for a week at 4℃. These data strongly suggested the complex is more suitable for crystallization. The complex was crystallized in vapor diffusion. We acquired some crystals of complex but their resolution had very low at the present study. So, we have been trying to crystallization in vapor diffusion and LCP under the various conditions. Less
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