Abstract
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.
Original language | English (US) |
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Pages (from-to) | 713-721 |
Number of pages | 9 |
Journal | Toxicon |
Volume | 41 |
Issue number | 6 |
DOIs | |
State | Published - May 1 2003 |
Externally published | Yes |
Keywords
- Anti-platelet aggregation effect
- Phospholipase A
- Recombinant expression
- Sea snake Lapemis hardwickii
- Site-directed mutagenesis
ASJC Scopus subject areas
- Toxicology