Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs.
BMC Biotechnol
; 11: 57, 2011 May 23.
Article
en En
| MEDLINE
| ID: mdl-21605442
ABSTRACT
BACKGROUND:
G protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability.RESULTS:
Here we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered ß2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (ß2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active ß2AR -T4L in cell-free systems. The binding specificity of the NLP- ß2AR-T4L complex has been determined by competitive assays. Our results demonstrate that ß2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor.CONCLUSIONS:
Although the activation of ß2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Receptores Adrenérgicos beta 2
/
Membrana Dobles de Lípidos
Límite:
Humans
Idioma:
En
Revista:
BMC Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Estados Unidos