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.
Pan
F M
FM
Institute of Biochemical Sciences, National Taiwan University and Institute of Biological Chemistry, Taipei.
Yeh
M S
MS
Chang
W C
WC
Hung
C C
CC
Chiou
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002038:Bungarotoxins / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004546:Elapid Venoms / Q000378:metabolism*; D017815:Elapidae / Q000235:genetics; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D054467:Phospholipases A2; D010957:Plasmids; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000737:chemistry; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013624:Taiwan
D002038:Bungarotoxins; D017931:DNA Primers; D004546:Elapid Venoms; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D010741:Phospholipases A; D054467:Phospholipases A2