| Project/Area Number |
15590122
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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 |
Medical pharmacy
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| Research Institution | Josai International University |
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Principal Investigator |
TOMIOKA Yoshihisa Josai International University, Department of Pharmaceutical Sciences, Professor, 薬学部, 教授 (00282062)
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| Co-Investigator(Kenkyū-buntansha) |
GOTO Junichi Tohoku University, University Hospital, Professor, 病院・教授 (80006337)
HISHINUMA Takanori Tohoku University, University Hospital, Associate Professor, 病院・助教授 (20199003)
OZAWA Mika Josai International University, Department of Pharmaceutical Sciences, Research Instructor, 薬学部, 助手 (40398558)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | phage display / neocarzinostatin / antibody-like protein / amino acid substitution / ethidium bromide / genetic library / megaprimer PCR mutagenesis / supernatural product / エチジウムブロミド |
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| Research Abstract |
In order to investigate the amino acid residues in apo-NCS involving the binding with the NCS-chr, the amino acid-substituted apo-NCS mutants (S98C, S98G, S98T and S98A) were prepared. The binding of the non-natural chromophore, EtdBr, with the each mutated apo-NCS was evaluated by the monitoring for the total fluorescence intensity and fluorescence polarization. The S98G mutant had the best binding property compared with another apo-NCS. The result suggested that the strategy of the substitution for the proper amino acid residues in apo-NCS could be expected to create supernatural apo-NCS. The randomized libraries were successfully prepared using the megaprimer PCR mutagenesis, the overlap extension PCR mutagenesis with dam methylase / DpnI digestion method. The mutated amino acids were total 14, and their sites were region A (49-52, PADF), region B (76-80, FLFDG), and region C (94-98, QVGLS), which are involving the binding with NCS-chr. The randomized library was successfully inserted into the vector DNA for the phage display, and performed a screening using bio-panning method against the glycyrrhetic acid conjugated bovine serum albumin (GA-BSA), and then isolated the clones which had a specific binding property to the GA-BSA. Hereafter, the detail structure analysis will be investigated for the isolated clones.
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