2005 Fiscal Year Final Research Report Summary
Development of a method for chemical synthesis of G-protein coupled receptor
| Project/Area Number |
14380287
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
AIMOTO Saburo Osaka University, Institute for Protein Research, Laboratory of Protein Organic Chemistry, Professor, 蛋白質研究所, 教授 (80029967)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAKAMI Toru Osaka University, Institute for Protein Research, Laboratory of Protein Organic Chemistry, Associate Professor, 蛋白質研究所, 助教授 (70273711)
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| Project Period (FY) |
2002 – 2005
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| Keywords | GPCR / nociceptin receptor / peptide thioester / chemical synthesis / coupling conditions / native chemical ligation / thioester method / auxiliary |
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| Research Abstract |
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
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