Diversity of cDNAs encoding phospholipase A 2 from Agkistrodon halys Pallas venom, and its expression in E. coli

Abstract

As a step toward understanding the structure and function of phospholipase A 2(PLA 2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA 2 isoenzymes including B-PLA 2, Asn 49-PLA 2, A-PLA 2, A′-PLA 2 and BA 1-PLA 2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes. The amino acid sequences of A-PLA 2 deduced from cDNA are consistent with that isolated from venom except for four residues. Asn 49–PLA 2 and B-PLA 2 are highly similar (>95%), but the critical residue Asp 49 in the active centre of B-PLA 2 is replaced by Asn 49 in Asn 49–PLA 2. The N-terminal residues (1–24) of BA1-PLA 2 shows high similarity to that of B-PLA 2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72–125) are the same as that of A-PLA 2 which can inhibit platelet aggregation. The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA 2s, but also to disclose the genesis of structural diversity of PLA 2s, namely DNA modification and gene rearrangement. The cloned cDNA for A-PLA 2 has been expressed in E. coli. By Q-Sepharose column chromatography, denaturation–renaturation and FPLC, we obtained the active recombinant protein with the initiator Met. This is the first report of the production of an active recombinant PLA 2 with the initiator Met.