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The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening

Paper ID Volume ID Publish Year Pages File Format Full-Text
25589 43583 2006 11 PDF Available
Title
The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening
Abstract

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was identified as a major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells. LOX-1 critically mediates the endothelial dysfunction and the progression of atherosclerosis by oxLDL stimulation. It might be an important target for vascular endothelium. In order to obtain human LOX-1 and identify its mimic ligand for facilitating the study of LOX-1 function, a recombinant plasmid pPIC9K-His-hLOX-1 was structured and expressed human LOX-1 in Pichia pastoris GS115. Western blot analysis ensured the expressed recombinant human LOX-1 protein and a receptor–ligand binding assay showed that it had high binding affinity with oxLDL. With this receptor protein, a competitive fluorescence polarization-based high throughput screening method was established in a 384-well microplate to isolate the mimic ligands of human LOX-1. The evaluating parameter Z′ value of 0.72 for this method showed that fluorescence polarization-based high throughput screening assay was robust and the results had a high reliability. By the fluorescence polarization-based high throughput screening assay, a total of 20,316 chemicals were screened, and 2 chemicals were identified that they have a high affinity with human LOX-1. Competitive uptake DiI–oxLDL assay by human LOX-1 transfected CHO-K1 cells further confirmed that two chemicals block the uptake of DiI–oxLDL. And the preliminary results indicated that isolated mimic ligands may act as a function of antagonist. The discovery of human LOX-1 mimic ligand would benefit to further study the function of LOX-1 and identify a novel avenue for prevention and treatment atherosclerosis.

Keywords
Lectin-like low-density lipoprotein receptor-1; Fluorescence polarization; High throughput screening; Atherosclerosis; Endothelial dysfunction
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The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 125, Issue 4, 1 October 2006, Pages 492–502
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us