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

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.

Additive or synergetic effects of phenolic compounds on human low density lipoprotein oxidation.

The in vitro assessment of the antioxidant capacity of four phenolic compounds; catechin, hesperidin, ferulic acid, and quercetin was evaluated by the examination of their ability to inhibit copper (Cu(2+))-mediated human low density lipoprotein (LDL) oxidation by using the thiobarbituric acid-reactive substances (TBARS) assay. Individually, the enrichment of LDLs with various concentrations of catechin, hesperidin, ferulic acid, and quercetin produced both antioxidant and prooxidant effects depending on enrichment concentrations of the polyphenolic compounds. Catechin and hesperidin had predominantly antioxidant effects (51.1%, 76.9%, respectively) while ferulic acid and quercetin had mostly prooxidant effects (166.4%; 191.8%, respectively) on LDL oxidation. However, when the mixture of the four phenolic compounds was used to enrich the LDLs, significant antioxidant capacity was demonstrated at all enrichment levels with a dose-response. Synergistic effects of the polyphenolic compounds as mixtures in preventing human LDL oxidation may reflect that nutritional advantages are found in the consumption of a variety of fruits and vegetables in preventing LDL oxidation and perhaps a host of cardiovascular diseases.