A rapid, tag-free way to purify functional GPCRs.
J Biol Chem
; 300(1): 105558, 2024 Jan.
Article
em En
| MEDLINE
| ID: mdl-38097184
ABSTRACT
G protein-coupled receptors (GPCRs) play diverse signaling roles and represent major pharmaceutical targets. Consequently, they are the focus of intense study, and numerous advances have been made in their handling and analysis. However, a universal way to purify GPCRs has remained elusive, in part because of their inherent instability when isolated from cells. To address this, we have developed a general, rapid, and tag-free way to purify GPCRs. The method uses short peptide analogs of the Gα subunit C terminus (Gα-CT) that are attached to chromatography beads (Gα-CT resin). Because the Gα-CT peptides bind active GPCRs with high affinity, the Gα-CT resin selectively purifies only active functional receptors. We use this method to purify both rhodopsin and the ß2-adrenergic receptor and show they can be purified in either active conformations or inactive conformations, simply by varying elution conditions. While simple in concept-leveraging the conserved GPCR-Gα-CT binding interaction for the purpose of GPCR purification-we think this approach holds excellent potential to isolate functional receptors for a myriad of uses, from structural biology to proteomics.
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01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transdução de Sinais
/
Receptores Acoplados a Proteínas G
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article