DIVERSITY OF CDNAS ENCODING PHOSPHOLIPASE A(2) FROM AGKISTRODON HALYSPALLAS VENOM, AND ITS EXPRESSION IN ESCHERICHIA-COLI

As a step toward understanding the structure and function of phospholi pase A(2)(PLA(2)), we isolated several novel cDNAs encoding Agkistrodo n 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 thes e 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 BA(1)-PLA(2) sh ows high similarity to that of B-PLA(2) which has strong ability to he molyze erythrocytes, while its C-terminal residues (72-125) are the sa me as that of A-PLA(2) which can inhibit platelet aggregation. The suc cessful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA(2)s, but also to disclose the genesis of structural diversity of PLA(2)s, namel y 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 recombinan t protein with the initiator Met. This is the first report of the prod uction of an active recombinant PLA(2) with the initiator Met. (C) 199 8 Elsevier Science Ltd. All rights reserved.