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

Polymerase chain reaction (PCR) was employed to amplify cDNAs construc ted from the poly(A)(+)RNA of venom glands in Taiwan cobras to facilit ate the cloning and sequencing of phospholipase A(2) (PLA(2)) gene. Th e 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-terminat ion method. Sequencing several clones containing about 0.5 kb DNA inse rts constructed a complete and unambiguous full-length reading frame o f 468 base pairs covering a precursor for phospholipase A(2) with a de duced mature protein sequence of 119 amino acids and a 27 amino-acid s egment of signal peptide. The sequenced major PLA(2) with pI 4.991 sho ws a high degree of sequence homology to those PLA(2) of the same or c losely-related genus. The deduced protein sequence allows us to correc t and resolve some discrepancy between the sequences determined by con ventional protein sequencing (Toxicon, 19, 141(1981)) and X-ray crysta llography (Science, 250, 1560(1990)). Expression of PLA(2) in E. coli vector generated a polypeptide which can cross-react with the antiseru m against the native and purified PLA(2) from the same cobra venom alb eit with a much lower activity. (C) 1994 Academic Press, Inc.