Purification, gene cloning and expression of an acidic phospholipase A2 from Agkistrodon shedaoensis Zhao.
A protein with the activity of phospholipase A(2) named asAPLA(2) was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column, Source S, and Mono Q FPLC. Its molecular weight was estimated to be 19 kD by SDS-PAGE, and its pI was about 3.5 by IEF analysis. It inhibited the platelet aggregation that was induced by 1 micromol/ L ADP, and the IC(50) was determined to be 6 micromol/L. Degenerating primer was designed and synthesized according to the N-terminal amino acid sequence of asAPLA(2). Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. Its molecular weight and the pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar software according to the deduced amino acid sequence. Then the gene was cloned into the expression plasmid pET-40b(+) and expressed in E. coli BL21(DE3). Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native enzyme.
Jin
Qian
Q
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for BiologicalSciences, the Chinese Academy of Sciences, Shanghai 200031, China. zhouyc@sunm.shcnc.ac.cn
Yang
Li-Xia
LX
Jiao
Hao-Mang
HM
Lu
Bin
B
Wu
Yu-Qun
YQ
Zhou
Yuan-Cong
YC
D017836:Agkistrodon / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007527:Isoenzymes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D007527:Isoenzymes; D010741:Phospholipases A; D054467:Phospholipases A2