The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening.
J Biotechnol
; 125(4): 492-502, 2006 Oct 01.
Article
em En
| MEDLINE
| ID: mdl-16735073
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Mimetismo Molecular
/
Receptores Depuradores Classe E
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Polarização de Fluorescência
/
Ligantes
Tipo de estudo:
Diagnostic_studies
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Evaluation_studies
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Screening_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article