Diversity of cDNAs encoding phospholipase A2 from Agkistrodon halys pallas venom, and its expression in E. coli.
As a step toward understanding the structure and function of phospholipase A2(PLA2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA2 isoenzymes including B-PLA2, Asn49-PLA2, A-PLA2, A'-PLA2 and BA1-PLA2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes. The amino acid sequences of A-PLA2 deduced from cDNA are consistent with that isolated from venom except for four residues. Asn49-PLA2 and B-PLA2 are highly similar (> 95%), but the critical residue Asp49 in the active centre of B-PLA2 is replaced by Asn49 in Asn49-PLA2. The N-terminal residues (1-24) of BA1-PLA2 shows high similarity to that of B-PLA2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72-125) are the same as that of A-PLA2 which can inhibit platelet aggregation. The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA2s, but also to disclose the genesis of structural diversity of PLA2s, namely DNA modification and gene rearrangement. The cloned cDNA for A-PLA2 has been expressed in E. coli. By Q-Sepharose column chromatography, denaturation-renaturation and FPLC, we obtained the active recombinant protein with the initiator Met. This is the first report of the production of an active recombinant PLA2 with the initiator Met.
Shanghai Institute of Biochemistry, Chinese Academy of Science, PR China.
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Pan
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Liu
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Ou-Yang
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Yang
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Zhou
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Li
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Wu
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003435:Crotalid Venoms / Q000201:enzymology*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003435:Crotalid Venoms; D018076:DNA, Complementary; D007527:Isoenzymes; D010743:Phospholipids; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2