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Bacterial expression and characterization of starfish phospholipase A2

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Title: Bacterial expression and characterization of starfish phospholipase A2
Authors: Kishimura, Hideki Browse this author →KAKEN DB
Ojima, Takao Browse this author →KAKEN DB
Hayashi, Kenji Browse this author
Nishita, Kiyoyoshi Browse this author
Keywords: Asterina pectinifera
Bacterial expression
Group I
Phospholipase A2
Substrate specificity
Surface loop
Issue Date: Mar-2001
Publisher: Elsevier Science Inc.
Journal Title: Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology
Volume: 128
Issue: 3
Start Page: 565
End Page: 573
Publisher DOI: 10.1016/S1096-4959(00)00349-3
PMID: 11250552
Abstract: 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.
Type: article (author version)
Appears in Collections:水産科学院・水産科学研究院 (Graduate School of Fisheries Sciences / Faculty of Fisheries Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 岸村 栄毅

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