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首页> 外文期刊>BMC Biotechnology >Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs
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Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs

机译：无细胞合成功能性G蛋白偶联受体与纳米级双层盘复合。

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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 β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo -expressed receptor. Conclusions Although the activation of β2AR-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.
机译：背景技术G蛋白偶联受体（GPCR）代表了人类基因组中最大的膜蛋白家族，是制药工业最丰富的靶标来源。表征GPCR的主要限制是难以开发经济高效的高级异源表达系统。这些困难的原因包括质膜运输和插入效率低下以及细胞毒性。此外，GPCR纯化需要使用去污剂，该去污剂会对受体的收率和稳定性产生负面影响。结果在这里我们报告了一种基于无洗涤剂的无细胞蛋白表达的方法，可在约2小时内获得具有药理活性的GPCR。我们的策略依赖于修饰的GPCR在纳米尺寸平面膜中的共翻译插入。作为模型，我们使用了工程化的β2-肾上腺素受体，其中第三个细胞内环已被T4溶菌酶（β2AR-T4L）取代。我们证明了纳米脂蛋白颗粒（NLPs）在无细胞系统中表达活性β2AR-T4L是必需的。 NLP-β2AR-T4L复合物的结合特异性已经通过竞争性测定法确定。我们的结果表明，体外合成的β2AR-T4L依赖于与体内表达的受体相似的氧化条件。结论尽管β2AR-T4L的激活需要插入T4溶菌酶序列，并且该活性蛋白的产量受到限制，但我们的结果从概念上证明了无细胞蛋白表达可以用作表达这些有价值且众所周知的困难的快速方法，表达蛋白质。

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