『徳島大学 教育・研究者情報データベース (EDB)』---[学外] /
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種別 (必須): 学術論文 (審査論文) [継承]
言語 (必須): 英語 [継承]
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カテゴリ (推奨):
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学究種別 (推奨):
組織 (推奨): 1.徳島大学.工学部.生物工学科.生物反応工学講座 [継承]
著者 (必須): 1. (英) Umetsu M. (日) (読)
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学籍番号 (推奨):
[継承]
2. (英) Tsumoto K. (日) (読)
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学籍番号 (推奨):
[継承]
3. (英) Hara M. (日) (読)
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学籍番号 (推奨):
[継承]
4. (英) Ashish K. (日) (読)
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5.郷田 秀一郎 ([長崎大学])
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6. (英) Adschiri T. (日) (読)
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[継承]
7. (英) Kumagai I. (日) (読)
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[継承]
題名 (必須): (英) How additives influence the refolfing of immunoglobulin-folded proteins in a stepwise dialysis system  (日)    [継承]
副題 (任意): (英) Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment  (日)    [継承]
要約 (任意): (英) The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and l-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 m GdnHCl to 1 m GdnHCl, and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt binding analysis indicated that the addition of l-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether l-arginine was present. Spontaneous folding at the 1 m GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of l-arginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of l-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether l-arginine was present. Controlled coupling of the effects of GSSG and l-arginine led to the complete refolding of scFv in the stepwise dialysis.  (日)    [継承]
キーワード (推奨): 1. (英) Anilino Naphthalenesulfonates (日) (読) [継承]
2. (英) Animals (日) (読) [継承]
3. (英) Circular Dichroism (日) (読) [継承]
4. (英) Dialysis (日) (読) [継承]
5. (英) Disulfides (日) (読) [継承]
6. (英) Dose-Response Relationship, Drug (日) (読) [継承]
7. (英) Escherichia coli (日) (読) [継承]
8. (英) Fluorescent Dyes (日) (読) [継承]
9. (英) Guanidine (日) (読) [継承]
10. (英) Immunoglobulin Fragments (日) (読) [継承]
11. (英) Immunoglobulin Variable Region (日) (読) [継承]
12. (英) Immunoglobulins (日) (読) [継承]
13. (英) Mass Spectrometry (日) (読) [継承]
14. (英) Mice (日) (読) [継承]
15. (英) Models, Chemical (日) (読) [継承]
16. (英) Protein Conformation (日) (読) [継承]
17. (英) Protein Folding (日) (読) [継承]
18. (英) Protein Structure, Secondary (日) (読) [継承]
19. (英) Protein Structure, Tertiary (日) (読) [継承]
20. (英) Spectrometry, Fluorescence (日) (読) [継承]
21. (英) Spectroscopy, Fourier Transform Infrared (日) (読) [継承]
22. (英) Tryptophan (日) (読) [継承]
発行所 (推奨):
誌名 (必須): The Journal of Biological Chemistry ([The American Society for Biochemistry and Molecular Biology])
(pISSN: 0021-9258, eISSN: 1083-351X)

ISSN (任意): 0021-9258
ISSN: 0021-9258 (pISSN: 0021-9258, eISSN: 1083-351X)
Title: The Journal of biological chemistry
Title(ISO): J Biol Chem
Publisher: American Society for Biochemistry and Molecular Biology
 (NLM Catalog  (Scopus  (CrossRef (Scopus information is found. [need login])
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(必須): 8979 8987 [継承]
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年月日 (必須): 西暦 2003年 3月 末日 (平成 15年 3月 末日) [継承]
URL (任意):
DOI (任意): 10.1074/jbc.M212247200    (→Scopusで検索) [継承]
PMID (任意): 12519771    (→Scopusで検索) [継承]
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WOS (任意): 000181524000016 [継承]
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備考 (任意): 1.(英) Article.Affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 07, Aoba-ku, Sendai, Japan.  (日)    [継承]
2.(英) Article.PublicationTypeList.PublicationType: Journal Article  (日)    [継承]
3.(英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't  (日)    [継承]

標準的な表示

和文冊子 ● M. Umetsu, K. Tsumoto, M. Hara, K. Ashish, Shuichiro Goda, T. Adschiri and I. Kumagai : How additives influence the refolfing of immunoglobulin-folded proteins in a stepwise dialysis system, --- Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment ---, The Journal of Biological Chemistry, Vol.278, No.11, 8979-8987, 2003.
欧文冊子 ● M. Umetsu, K. Tsumoto, M. Hara, K. Ashish, Shuichiro Goda, T. Adschiri and I. Kumagai : How additives influence the refolfing of immunoglobulin-folded proteins in a stepwise dialysis system, --- Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment ---, The Journal of Biological Chemistry, Vol.278, No.11, 8979-8987, 2003.

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