Cloning, expression, and purification of lipoprotein-associated phospholipase A(2) in Pichia pastoris.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery. Here, we cloned the Lp-PLA(2) gene from differentiated THP-1 cells, and inserted a carboxy-terminal His(6)-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA(2) fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity of our recombinant Lp-PLA(2) was strong when [3H] PAF was used as a substrate, and the Lp-PLA(2) inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA2 (IC50 = 15.93 +/- 1 microM). This novel recombinant Lp-PLA(2) could prove useful as a screening model for Lp-PLA(2) inhibitors, and may facilitate further investigation of this protein in atherosclerosis.