Bacterial expression and characterization of starfish phospholipase A(2).
Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) 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 PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) 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 PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.
Kishimura
H
H
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. kishi@fish.hokudai.ac.ajp
Ojima
T
T
Hayashi
K
K
Nishita
K
K
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D005822:Genetic Vectors; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000032:analysis; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins; D013215:Starfish / Q000201:enzymology*; D013379:Substrate Specificity; D013997:Time Factors
D008055:Lipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010741:Phospholipases A; D002118:Calcium