Journal
BMC BIOTECHNOLOGY
Volume 11, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/1472-6750-11-57
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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 beta 2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (beta 2AR-T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active beta 2AR-T4L in cell-free systems. The binding specificity of the NLP-beta 2AR-T4L complex has been determined by competitive assays. Our results demonstrate that beta 2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor. Conclusions: Although the activation of beta 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.
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