1. Heterologous expression of lipoprotein-associated phospholipase A2 in different expression systems.
- Author
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Zhang F, Dong L, Cai M, Shen J, and Wang Y
- Subjects
- Animals, Escherichia coli genetics, Insecta cytology, Insecta genetics, Phospholipases A isolation & purification, Phospholipases A metabolism, Phospholipases A2, Pichia genetics, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Gene Expression, Lipoproteins, LDL metabolism, Phospholipases A biosynthesis, Phospholipases A genetics
- Abstract
Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a key enzyme involved in atherosclerosis, and has been considered as a new target for drug discovery. The major difficulty for high-throughput screening of Lp-PLA(2) inhibitors and for functional studies was their fast and efficient production. Purification of native Lp-PLA(2) from human plasma was complicated and produced a very low yield. We herein examined the feasibility of expressing and purifying recombinant Lp-PLA(2) in different heterologous expression systems. The fusion Lp-PLA(2) was expressed at high levels and exhibited strong enzyme activity in insect cell-baculovirus expression system. The functional enzyme could also be produced in Pichia pastoris. The inclusion of a Kozak sequence increased greatly the expression level of recombinant Lp-PLA(2) in insect cells, but had little effect on the expression of recombinant Lp-PLA(2) in P. pastoris and Escherichia coli. P. pastoris-produced Lp-PLA(2) could be purified rapidly and conveniently through a one-step procedure, while baculovirus-produced Lp-PLA(2) could be efficiently purified through a two-step procedure. This ability to readily produce recombinant Lp-PLA(2) could provide a screening model for Lp-PLA(2) inhibitors and will facilitate further studies on this enzyme.
- Published
- 2006
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