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.
Zhang
Fujun
F
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Graduate School, Chinese Academy of Sciences, Shanghai 201203, China.
Wang
Yiping
Y
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D010843:Pichia / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase