Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs.

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 ?2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (?2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active ?2AR -T4L in cell-free systems. The binding specificity of the NLP- ?2AR-T4L complex has been determined by competitive assays. Our results demonstrate that ?2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor. CONCLUSIONS: Although the activation of ?2AR-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.

SUBMITTER: Yang JP 

PROVIDER: S-EPMC3125327 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature