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Methods Mol Biol ; 1272: 173-88, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25563184

RESUMO

The G protein-coupled receptors (GPCRs) form one of the largest membrane receptor families. The nature of the ligands that interact with these receptors is highly diverse; they include light, peptides and hormones, neurotransmitters, and small molecular weight compounds. The GPCRs are involved in a wide variety of physiological processes and thus hold considerable therapeutic potential.GPCR function is usually determined in cell-based assays, whose complexity nonetheless limits their use. The use of alternative, cell-free assays is hampered by the difficulties in purifying these seven-transmembrane domain receptors without altering their functional properties. Several methods have been proposed to immobilize GPCR on biosensor surfaces which use antibodies or avidin-/biotin-based capture procedures, alone or with reconstitution of the GPCR physiological microenvironment. Here we propose a method for GPCR immobilization in their native membrane microenvironment that requires no manipulation of the target receptor and maintains the many conformations GPCR can adopt in the cell membrane.


Assuntos
Vetores Genéticos/metabolismo , Proteínas Imobilizadas/genética , Lentivirus/genética , Plasmídeos/metabolismo , Receptores CXCR4/genética , Vesiculovirus/genética , Carbocianinas , Corantes Fluorescentes , Expressão Gênica , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Imobilizadas/metabolismo , Lentivirus/metabolismo , Plasmídeos/química , Receptores CXCR4/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Transfecção , Vesiculovirus/metabolismo , Vírion , Montagem de Vírus/genética
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