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著作: Ishido Tomomi/[山﨑 尚志]/[石川 満]/Hirano Ken/Characterization of DNA polymerase from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid./[Microbial Cell Factories]

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EID
296077
EOID
802145
Map
0
LastModified
2015年11月11日(水) 20:24:04
Operator
大家 隆弘
Avail
TRUE
Censor
0
Owner
山﨑 尚志
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種別 必須 学術論文(審査論文)
言語 必須 英語
招待 推奨
審査 推奨
カテゴリ 推奨 研究
共著種別 推奨
学究種別 推奨
組織 推奨
著者 必須
  1. (英) Ishido Tomomi
    役割 任意
    貢献度 任意
    学籍番号 推奨
  2. 山﨑 尚志([徳島大学.大学院医歯薬学研究部.薬学域.薬科学部門.生命薬学系.薬物治療学])
    役割 任意
    貢献度 任意
    学籍番号 推奨
  3. 石川 満([独立行政法人 産業技術総合研究所])
    役割 任意
    貢献度 任意
    学籍番号 推奨
  4. (英) Hirano Ken
    役割 任意
    貢献度 任意
    学籍番号 推奨
題名 必須

(英) Characterization of DNA polymerase from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid.

副題 任意
要約 任意

(英) Eukaryotic DNA polymerase β (pol β), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. However, pol β has not been purified or enzymatically characterized from the model fish species Danio rerio (zebrafish). We used the in vitro/in vivo dual expression system plasmid, pIVEX, to express Danio rerio pol β (Danio pol β) for biochemical characterization. Danio pol β encoded by the in vitro/in vivo-compatible pIVEX plasmid was expressed in E. coli BL21(DE3), BL21(DE3)pLysS, and KRX, and in vitro as a C-terminal His-tagged protein. Danio pol β expressed in vitro was subject to proteolysis; therefore, bacterial overexpression was used to produce the protein for kinetic analyses. KRX cells were preferred because of their reduced propensity for leaky expression of pol β. The cDNA of Danio rerio pol β encodes a protein of 337 amino acids, which is 2-3 amino acids longer than other pol β proteins, and contains a P63D amino acid substitution, unlike mammalian pol βs. This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. We performed extensive biochemical characterization of Danio pol β in comparison with rat pol β, which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30°C). Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (kcat/Km). Our characterization of pol β from a model fish organism contributes to the study of the function and evolution of DNA polymerases, which are emerging as important cellular targets for chemical intervention in the development of anticancer agents.

キーワード 推奨
  1. (英) Amino Acid Sequence
  2. (英) Animals
  3. (英) DNA Polymerase beta
  4. (英) Enzyme Stability
  5. (英) Escherichia coli
  6. (英) Fish Proteins
  7. (英) Gene Expression
  8. (英) Kinetics
  9. (英) Molecular Sequence Data
  10. (英) Plasmids
  11. (英) Protein Structure, Tertiary
  12. (英) Rats
  13. (英) Recombinant Fusion Proteins
  14. (英) Sequence Homology, Amino Acid
  15. (英) Zebrafish
発行所 推奨 BioMed Central Ltd.
誌名 必須 Microbial Cell Factories([BioMed Central Ltd.])
(pISSN: 1475-2859)
ISSN 任意 1475-2859
ISSN: 1475-2859 (eISSN: 1475-2859)
Title: Microbial cell factories
Title(ISO): Microb Cell Fact
Publisher: BioMed Central
 (NLM Catalog  (Scopus  (CrossRef (Scopus information is found. [need login])
必須 10
必須 ---
必須 84 84
都市 任意
年月日 必須 2011年 10月 21日
URL 任意
DOI 任意 10.1186/1475-2859-10-84    (→Scopusで検索)
PMID 任意 22018137    (→Scopusで検索)
NAID 任意
WOS 任意
Scopus 任意
評価値 任意
被引用数 任意
指導教員 推奨
備考 任意
  1. (英) Article.ELocationID: 10.1186/1475-2859-10-84

  2. (英) Article.PublicationTypeList.PublicationType: Journal Article

  3. (英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't

  4. (英) OtherID: PMC3214136