Expression, purification and biochemical characterization of a recombinant phospholipase A2, with anticoagulant activity from Agkistrodon halys Pallas.
A cloned cDNA encoding a PLA2 from Agkistrodon halys Pallas was found to have conservative residues Glu53 and Trp70 but with Lys56 and Lys67 substituted by Thr56 and Asp67, respectively, when compared with sequences of other class II PLA2 with anticoagulant activity. It was inserted into a temperature-sensitive bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies and recovered by centrifugation after enzyme digestion. By washing to partial purification, the expression product was refolded and was purified by FPLC superose 12 to appear as a single band in SDS-PAGE. The recombinant protein proved to have obvious enzymatic, anticoagulant and hemolytic activities, which were removed after modification by p-BPB. These findings suggest that the pharmacological activities of this recombinant PLA2 may be related to its catalytic activity and warrant further research on the structure-function relationships of the pharmacological site of the PLA2 from Agkistrodon halys Pallas.
Zhong
X
X
Shanghai Institute of Biochemistry, Chinese Academy of Sciences.
Liu
J
J
Wu
X
X
Zhou
Y
Y
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D003435:Crotalid Venoms / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005822:Genetic Vectors; D006461:Hemolysis; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000494:pharmacology
C031442:Agkistrodon venoms; D000925:Anticoagulants; D003435:Crotalid Venoms; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2