Sequence analysis and expression of phospholipase A₂ 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 A₂ (PLA₂) 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 A₂ with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid segment of signal peptide. The sequenced major PLA₂ with pI 4.991 shows a high degree of sequence homology to those PLA₂ 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 PLA₂ in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified PLA₂ from the same cobra venom albeit with a much lower activity.