Refolding Experiment Using "Loose Folding Method" That Induces Correct 3D Structure from Denatured Protein
| Project/Area Number |
09672194
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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 |
Physical pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
SAKAI Tomoya Nagoya City University, Faculty of Pharmaceutical Sciences, Department of Chemical Reaction Engineering, Professor, 薬学部, 教授 (00080169)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Ei-ichiro Central Research Laboratories, Ajinomoto Co., Inc., Senior Chief Scientist, 中研, 主席研究員
KURIMOTO Eiji Nagoya City University, Faculty of Pharmaceutical Sciences, Department of Chemic, 薬学部, 助手 (90234575)
KURODA Yoshitaka Nagoya City University, Faculty of Pharmaceutical Sciences, Department of Chemic, 薬学部, 講師 (40080204)
NOHARA Daisuke Nagoya City University, Faculty of Pharmaceutical Sciences, Department of Chemic, 薬学部, 助教授 (60080214)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1998: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Protein / Refolding / Loose folding / ル-スフォールディング |
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| Research Abstract |
(1) Refolding experiment was carried out by use of denatured and reduced hen egg-white lysozyme (Lyzm). Recovered activity was ca. 20% at the Lyzm concentration of 10 muM by simple dilution method from 6 M guanidinium chloride (GdmCl) solution. The major part of the unrecovered enzyme resulted in aggregation. In the presence of 0.5-1 M GdmCl where the protein molecule is expected to be in loosely refolded state, the recovered activity increased drastically up to 80%. Using combined solvent of urea and LiCl instead of GdmCl, the recovered activity of 100% could be attained.
(2) Combined solvents such as ethylene glycol-LiCl and glycerol-LiCl also brought about the perfect recovery of the denatured and reduced Lyzm probably due to realization of the loosely refolded state of protein molecule.
(3) For the refolding of Streptomyces griseus trypsin (SGT) the solution of triethanolamine or diethanolamine at 1-2 M revealed to be excellent medium whereas urea-LiCl or potassium acetate effective to the refolding of Lyzm or subtilisin BPN', respectively was not useful to the refolding of SGT.Furthermore, it was found that for the refolding of the protease such an additive device as prevention of autolysis guaranteed the increase of the recovery yield beside the selection of refolding media to induce the loosely refolded state of denatured protein molecules.
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Report
(3 results)
Research Products
(15 results)
