Functional expression and characterization of a recombinant phospholipase A₂ from sea snake Lapemis hardwickii as a soluble protein in E. coli

Three full-length phospholipase A₂ (PLA₂) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA₂-9 in E. coli. The open reading frame encoding mature peptide of PLA₂-9 was subcloned into the vector pTRX. The Trx-PLA₂-9 fusion protein was expressed as a soluble protein by IPTG induction at 23°C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA₂-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA₂-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA₂-9 (rPLA₂-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA₂, which shares 90% nucleotide sequence similarity to PLA₂-9, the rPLA₂-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA₂-9 may be dissociated.