Development of a method for chemical synthesis of G-protein coupled receptor

为了建立一种g蛋白偶联受体（gpcr）的合成方法，我们对膜蛋白合成所需的因子进行了全面的研究。我们选择了1类阿片受体的c端区域ORL1（251-370）作为靶分子，它包含第6和第7跨膜区域以及c端胞质结构域。ORL1（251-370）被分成三个肽段用于合成。每个肽段采用固相法合成。含有跨膜区域的肽硫酯在HPLC纯化的溶剂中几乎不溶。在硫酯的硫基部分引入五精氨酸标签增强了肽硫酯的溶解度。用反相高效液相色谱法对肽段进行纯化。含有第七跨膜区域的纯化肽硫酯通过天然化学连接与c端结构域缩合。在优化的条件下，以洗涤剂的浓度、缓冲液的pH、硫醇化合物作为添加剂等为条件，几乎定量地进行了结扎。然而，含有第6跨膜区域的肽硫酯与含有第7跨膜区域和c端区域的肽之间的第二次偶联是不成功的。没有找到合适的保护组。然后，我们开发了两种结扎辅助剂，允许在没有侧链保护的情况下进行节段耦合。通过光照射去除辅助剂，这是膜蛋白合成的理想特性。ORL1（251-370）正在使用这些助剂进行再合成。我们还开发了一种制备肽硫酯的新方法，该方法是在形成硫酯的c端氨基酸上自由再电离。本研究的发现将对阐明膜蛋白的结构和功能有重要意义。少

In order to develop a synthetic method for G-protein-coupled receptors (GPCRs), we performed comprehensive studies on the factors which would be required for membrane protein synthesis. We chose the C-terminal region of opioid receptor like 1, ORL1(251-370), as a target molecule that contained the sixth and seventh transmembrane regions and the C-terminal cytosolic domain. The ORL1(251-370) was divided into three peptide segments for synthetic purpose. Each peptide segment was synthesized by the solid phase method. Peptide thioesters that contained a transmembrane region were hardly soluble in solvents used for HPLC purification. Introduction of a penta-arginine tag into a thiol moiety in the thioester enhanced the solubility of the peptide thioesters. Then the peptide segment was purified by reversed-phase HPLC much easier than before. The purified peptide thioester that contained the seventh transmembrane region was condensed with the C-terminal domain by the native chemical ligation … More . The ligation proceeded almost quantitatively under the optimized conditions in terms of the concentration of a detergent, pH of a buffer, a thiol compound as an additive and etc. However, the second coupling between a peptide thioester that contained the sixth transmembrane region and the peptide that contained the seventh transmembrane region and the C-terminal region was unsuccessful. No appropriate protecting groups were found. Then, we developed two ligation auxiliaries that permitted segment coupling without side chain protections. The auxiliaries are removed by photo-irradiation and this is ideal characteristics for membrane protein synthesis. The ORL1(251-370) is under resynthesized using these auxiliaries. We also developed a novel method for the preparation of peptide thioesters, which were free recemization at the C-terminal amino acid that formed thioester. The findings obtained through this research will greatly contribute to elucidate the structure and function of membrane proteins. Less