Acta Pharmacologica Sinica 2006 June; 27 (6): 679-684; doi: 10.1111/j.1745-7254.2006.00308.x

Aim: To express and purify lipoprotein-associated phospholipase A₂ (Lp-PLA₂), and to establish a screening model for Lp-PLA₂ inhibitors using the expressed Lp-PLA₂.

Methods: We cloned the full-length cDNA of Lp-PLA₂ 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 Ni²⁺ affinity chromatography. Recombinant Lp-PLA₂ activity was measured using [³H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA₂ pretreated with the known Lp-PLA₂ inhibitor SB435495.

Results: We successfully cloned the full-length Lp-PLA₂ 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-PLA₂ activity was measured using [³H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA₂ inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA₂ with an IC₅₀ of 57±1 µmol/L.

Conclusion: We expressed and purified Lp-PLA₂ 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-PLA₂ inhibitors using the expressed Lp-PLA₂.

Keywords: lipoprotein-associated phospholipase A₂; atherosclerosis; coronary heart diseases; cloning; baculovirus; purification