| Project/Area Number |
17360399
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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 | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | OKYAMA UNIVERSITY |
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Principal Investigator |
YAMADA Hidenori OKYAMA UNIVERSITY, Graduate school of natural science and technology, professor, 大学院自然科学研究科, 教授 (80037613)
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| Co-Investigator(Kenkyū-buntansha) |
FUTAMI Junichiro OKYAMA UNIVERSITY, Graduate school of natural science and technology, Lecturer, 大学院自然科学研究科, 講師 (00420498)
SENO Masaharu OKYAMA UNIVERSITY, Graduate school of natural science and technology, professor, 大学院自然科学研究科, 教授 (90243493)
TADA Hiroko OKYAMA UNIVERSITY, Graduate school of natural science and technology, Assistant, 大学院自然科学研究科, 助手 (60271061)
KOSAKA Megumi Advanced Science Research Center, Assistant, 自然生命科学研究支援センター, 助手 (00170233)
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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 |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2006: ¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 2005: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Keywords | Protein / chemical modification / protein transduction / folding / protein purfication / cell culture / protein engineering / biotechnology |
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| Research Abstract |
This project aimed for the development of two methodologies; first is efficient solubilization of denatured proteins with reversible protein chemical cationization techniques, and second is intracellular delivery of reversibly cationized proteins followed by simultaneous folding to the active conformations, called in cell folding techniques. As a model protein, we investigated human tumor-suppressor p53. Bacterially expressed p53 protein as insoluble inclusion body was successfully solubilized by introduction of polyethylenimine 600 (PEI600, molecular weight: 600) via disulfide bond. The analysis of resulted PEI600-SS-p53 revealed that six cysteins were conjugated by PEI600, but remaining four cysteins failed in conjugation of PEI600, probably because of steric hindrance. This result indicated that reversible blocking of remaining free sulfidryl groups by a small molecule of aminopropyl methanethiosulfonate is important for preparation of chemically stable samples. The 'in cell folding' of p53 was successfully demonstrated by treatment of p53-null Saos-2 cells with reversibly cationized p53. PEI600-SS-p53 treated cells revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. Cationic charge dependent protein transduction efficiency was seen on the comparison between APS-SS-p53 (net charge: +6) and PEI600-SS-p53 (net charge: +81.6) because more cationic PEI600-SS-p53 showed more efficient induction of p53 targeted gene expression. This project also revealed that most of denatured proteins possessing cysteine residues can be solubilized by introduction of excess cationic charges. Furthermore, protein transduction and induction of their biological functions in living cells by using in cell folding techniques were confirmed by using at least 6 kinds of proteins.
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