Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli.
Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E. coli. The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX. The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.
Yang
Wen-Li
WL
The Open Laboratory for Marine Functional Genomics of State High-Tech Development, Department of Biochemistry, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, People's Republic of China.
Peng
Li-Sheng
LS
Zhong
Xiao-Fen
XF
Wei
Jian-Wen
JW
Jiang
Xiao-Yu
XY
Ye
Lan-Ting
LT
Zou
Lan
L
Tu
Hong-Bin
HB
Wu
Wen-Yan
WY
Xu
An-long
AL
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects; D018076:DNA, Complementary / Q000302:isolation & purification; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D009154:Mutation / Q000235:genetics; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D054467:Phospholipases A2; D010974:Platelet Aggregation / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility
D018076:DNA, Complementary; D004546:Elapid Venoms; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2