Expression and purification of lipoprotein-associated phospholipase A(2), a key enzyme involved in atherosclerosis.

AIM

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.

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-PL A(2) inhibitors using the expressed Lp-PLA(2).