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Bacterial expression and characterization of starfish phospholipase A2

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タイトル: Bacterial expression and characterization of starfish phospholipase A2
著者: Kishimura, Hideki 著作を一覧する
Ojima, Takao 著作を一覧する
Hayashi, Kenji 著作を一覧する
Nishita, Kiyoyoshi 著作を一覧する
キーワード: Asterina pectinifera
Bacterial expression
Group I
Phospholipase A2
Substrate specificity
Surface loop
発行日: 2001年 3月
出版者: Elsevier Science Inc.
誌名: Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology
巻: 128
号: 3
開始ページ: 565
終了ページ: 573
出版社 DOI: 10.1016/S1096-4959(00)00349-3
抄録: Phospholipase A2 (PLA2) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA2 from porcine pancreas. To investigate enzymatic properties of the starfish PLA2 in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA2 cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA2 protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-β-(–)-thiogalactopyranoside. The recombinant PLA2 produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris–HCl buffer (pH 8.0). Renatured PLA2 was purified by subsequent column chromatographies on DEAE–cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA2 was replaced by an Ala in the recombinant PLA2, the recombinant enzyme showed essentially the same properties as did the native PLA2 with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca2+ requirement.
Relation (URI):
資料タイプ: article (author version)
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 岸村 栄毅


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