SEQUENCE-ANALYSIS AND EXPRESSION OF PHOSPHOLIPASE A(2) FROM TAIWAN COBRA

Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)(+)RNA of venom glands in Taiwan cobras to facilitate the cloning and sequencing of phospholipase A(2) (PLA(2)) gene. The PCR product was then subcloned into pUC18 vector and transformed in E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing about 0.5 kb DNA inserts constructed a complete and unambiguous full-length reading frame of 468 base pairs covering a precursor for phospholipase A(2) with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid segment of signal peptide. The sequenced major PLA(2) with pI 4.991 shows a high degree of sequence homology to those PLA(2) of the same or closely-related genus. The deduced protein sequence allows us to correct and resolve some discrepancy between the sequences determined by conventional protein sequencing (Toxicon, 19, 141(1981)) and X-ray crystallography (Science, 250, 1560(1990)). Expression of PLA(2) in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified PLA(2) from the same cobra venom albeit with a much lower activity. (C) 1994 Academic Press, Inc.