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 571 mol/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.

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