Open AccessExpression and purification of lipoprotein‐associated phospholipase A 2 , a key enzyme involved in atherosclerosis 1
Author(s) -
ZHANG Fujun,
CAI Maojun,
SHEN Jingkang,
WANG Yiping
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00308.x
Subject(s) - sf9 , lipoprotein associated phospholipase a2 , recombinant dna , complementary dna , microbiology and biotechnology , phospholipase a2 , enzyme , affinity chromatography , biology , biochemistry , fusion protein , expression vector , enzyme assay , gene , spodoptera
Abstract Aim: To express and purify lipoprotein‐associated phospholipase A2 (Lp‐PLA 2 ), and to establish a screening model for Lp‐PLA 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 IC50 of 57±1 umol/L. Conclusion: 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‐PLA 2 inhibitors using the expressed Lp‐PLA 2 .