Bacterial expression and characterization of starfish phospholipase A 2

Abstract

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-β- 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.