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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
  • 1
    Citations
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    Usage
  • 13
    Captures
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    Mentions
  • 0
    Social Media
Metric Options:   Counts1 Year3 Year

Metrics Details

  • Citations
    1
    • Citation Indexes
      1
  • Captures
    13

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.

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