@article{APS3931,
author = {Fu-jun Zhang and Mao-jun Cai and Jing-kang Shen and Yi-ping Wang},
title = {Expression and purification of lipoprotein-associated phospholipase A 2 , a key enzyme involved in atherosclerosis},
journal = {Acta Pharmacologica Sinica},
volume = {27},
number = {6},
year = {2016},
keywords = {},
abstract = {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 57±1 μmol/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.},
issn = {1745-7254}, url = {http://www.chinaphar.com/article/view/3931}
}