| Project/Area Number |
12680744
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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 |
Neurochemistry/Neuropharmacology
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| Research Institution | Iwate university |
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Principal Investigator |
ORURUSU Tarou Iwate univ., engineering, professor, 工学部, 教授 (70177202)
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| Co-Investigator(Kenkyū-buntansha) |
SAKATA Kazumi Iwate univ., engineering, Research associate, 工学部, 助手 (80261163)
SHINGAI Ryuzo Iwate univ., engineering, professor, 工学部, 教授 (00089088)
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| Project Period (FY) |
2000 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | GABA / GABAc / rho subrunit / fusion protein / ヒトゲノム / GABA受容体ρサブユニット / 大腸菌発現系 |
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| Research Abstract |
1 GABA Receptor rho Subunits in Human Genome
GABA receptor rho subunits were explored in human geome by PCR and bioinformatics. There were rho1, rho2, rho3 and one pseudo gene.
2 Construction, Expression, and Characterization of rho/GABAa-chimera subunits in Xenopus oocytes.
3 Expression and Characterization of the N-Terminal Domain of Rat GABA Receptor rho3 Subunit. GABA receptor rho3 subunits form homooligomeric receptors in Xenopus oocytes. To explore the molecular mechanism underlying subunit assembly, the N-terminal domain of rho3 subunits were created and characterized. The DNA fragment corresponding to the N-terminal domain of rho3 subunit (242 aa) was subcloned into the maltose-binding protein vector (pMalc2, NEB) so that the coding sequence was fused in-frame to the 3' end of the malE gene. E coli cells bearing the fusion plasmid were grown, the tac promoter was induced with 0.4 mM IPTG, and the cells were harvested. A crude cell extract was prepared by sonication and passed over a column containing a cross-linked matrix of amylose (NEB). The fusion protein was eluted with 10 mM maltose and was cut with factore Xa to separate the N-terminal domain of rho3 subunit from maltose-binding protein. Sephacryl S-500 gel filtration chlomatography revealed that the N-terminal domain formed large assembly (500〜700 kDa) in the presence of 1 M NaCl, 10 mM GABA, and 0.1% tween-20. In 0.1 M NaCl, a small portion of the N-terminal domain was observed at 30〜60 kDa.
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