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Aim:To express and purify lipoprotein-associated phospholipase A_2(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-PLA2 from differentiatedTHP-I cells,and subcloned the cDNA into the baculovirus transfer vectorpFastBacl.In addition,we introduced an N-terminal Kozak sequence for high-level translation initiation and a C-terminal sequence of 6 histidine residues forpurification,The fusion enzyme was expressed in Sf9 insect cells and purified byNi~(2+)affinity chromatography.Recombinant Lp-PLA_2 activity was measured us-ing[~3H]PAF as a substrate,and we examined the enzyme activity of recombinantLp-PLA_2 pretreated with the known Lp-PLA_2 inhibitor SB435495.Results:Wesuccessfully 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 purifiedefficiently through a 2-step procedure.The recombinant Lp-PLA_2 activity wasmeasured using[~3H]PAF as a substrate,and proved to be enzymatically active.Lp-PLA_2 inhibitor SB435495 produced a good inhibition curve for inhibition ofrecombinant Lp-PLA_2 with an IC_(50)of 57±1μmol/L.Conclusion:We expressed andpurified Lp-PLA_2 at a high level in insect cell-baculovirus expression system.Theyield ratio was much greater than that obtained from human plasma and we estab-lished a screening model for Lp-PLA_2 inhibitors using the expressed Lp-PLA_2.
Aim: To express and purify lipoprotein-associated phospholipase A_2 (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-PLA2 from differentiated THP -I cells, and subcloned the cDNA into the baculovirus transfer vector pFastBacl.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+ -adsorption chromatography.Recombinant Lp-PLA_2 activity was measured by us- ing [~ 3H] 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: Wesuccessfully 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 purifielyly through a 2-step procedure. The recombinant Lp-PLA_2 activi L was used 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 μmol / L. Conlusion: We expressed and purified Lp-PLA_2 at a high level in insect cell-baculovirus expression system. The ratio was much greater than that obtained from human plasma and we estab-lished a screening model for Lp-PLA_2 inhibitors using the expressed Lp-PLA_2.