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

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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