Cloning, expression, and purification of lipoprotein-associated phospholipase A2 in Pichia pastoris

摘要

Lipoprotein-associated phospholipase A2 (Lp-PLA2) 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 His6-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA2 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-PLA2 was strong when [3H] PAF was used as a substrate, and the Lp-PLA2 inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA2 (IC50=15.93±1 μM). This novel recombinant Lp-PLA2 could prove useful as a screening model for Lp-PLA2 inhibitors, and may facilitate further investigation of this protein in atherosclerosis.