Expression and purification of lipoprotein-associated phospholipase A , a key enzyme involved in atherosclerosis
Acta Pharmacologica Sinica, ISSN: 1671-4083, Vol: 27, Issue: 6, Page: 679-684
2006
- 1Citations
- 13Captures
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Article Description
Aim: To express and purify lipoprotein-associated phospholipase A (Lp-PLA), and to establish a screening model for Lp-PLA inhibitors using the expressed Lp-PLA. Methods: We cloned the full-length cDNA of Lp-PLA 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 Ni affinity chromatography. Recombinant Lp-PLA activity was measured using [H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA pretreated with the known Lp-PLA inhibitor SB435495. Results: We successfully cloned the full-length Lp-PLA 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-PLA activity was measured using [H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA with an IC of 57±1 μmol/L. Conclusion: We expressed and purified Lp-PLA 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-PLA inhibitors using the expressed Lp-PLA. ©2006 CPS and SIMM.
Bibliographic Details
http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=33745360343&origin=inward; http://dx.doi.org/10.1111/j.1745-7254.2006.00308.x; http://www.ncbi.nlm.nih.gov/pubmed/16723085; https://www.nature.com/doifinder/10.1111/j.1745-7254.2006.00308.x; https://dx.doi.org/10.1111/j.1745-7254.2006.00308.x; https://www.nature.com/articles/aps200690; http://sciencechina.cn/gw.jsp?action=cited_outline.jsp&type=1&id=2462199&internal_id=2462199&from=elsevier
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