Expression and purification of lipoprotein-associated phospholipase A(2), a key enzyme involved in atherosclerosis.
OBJECTIVE
To express and purify lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), and to establish a screening model for Lp-PL A(2) inhibitors using the expressed Lp-PLA(2).


METHODS
We cloned the full-length cDNA of Lp-PLA(2) from differentiated THP-1 cells, and subcloned the cDNA into the baculovirus transfer vector pFastBac1. In addition, we introduced an N-terminal Kozak sequence for high-level translation initiation and a C-terminal sequence of 6 histidine residues for purification. The fusion enzyme was expressed in Sf9 insect cells and purified by Ni(2+) affinity chromatography. Recombinant Lp-PLA(2) activity was measured using [(3)H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA(2) pretreated with the known Lp-PLA(2) inhibitor SB435495.


RESULTS
We successfully cloned the full-length Lp-PLA(2) gene from differentiated THP-1 cells. The fusion enzyme was expressed in Sf9 insect cells at a high level and purified efficiently through a 2-step procedure. The recombinant Lp-PLA(2) activity was measured using [(3)H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA(2) inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA(2) with an IC(50) of 57+/-1 micromol/L.


CONCLUSIONS
We expressed and purified Lp-PLA(2) at a high level in insect cell-baculovirus expression system. The yield ratio was much greater than that obtained from human plasma and we established a screening model for Lp-PL A(2) inhibitors using the expressed Lp-PLA(2).
Zhang
Fu-Jun
FJ
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Cai
Mao-Jun
MJ
Shen
Jing-Kang
JK
Wang
Yi-Ping
YP
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D050197:Atherosclerosis / Q000201:enzymology*; D016367:Baculoviridae / Q000235:genetics; D001713:Biphenyl Compounds / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007948:Leukemia, Monocytic, Acute / Q000473:pathology; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011744:Pyrimidinones / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000235:genetics
D001713:Biphenyl Compounds; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011744:Pyrimidinones; D011993:Recombinant Fusion Proteins; C470971:SB-435495; D006639:Histidine; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase