| Project/Area Number |
62570965
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Physical pharmacy
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| Research Institution | Kyoto University |
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Principal Investigator |
NAKAGAWA Terumichi Faculty of Pharmaceutical Sciences,, 薬学部, 助教授 (70025708)
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| Co-Investigator(Kenkyū-buntansha) |
SHIBUKAWA Akimasa Faculty of Pharmaceutical Sciences, 薬学部, 助手 (30170913)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Membrane protein / Purification / Affinity chromatography / Size exclusion chromatography / Growth hormone / Growth hormone receptor / Membrane receptor / グルタルアルデヒドオリゴマー / 膜タンパク質の迅速精製システム / グルタルアルデヒド / グルタルアルデヒドポリマー / 固定化タンパク質 / 架橋剤 / 膜タンパク質 / 膜タンパク質の分離精製 / 受容体の高速分離精製 |
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| Research Abstract |
A high-performance liquid chromatographic system has been developed for the rapid purification of membrane proteins. The system was set up with an affinity chromatography and a size exclusion chromatography. The affinity gels were prepared by covalent bonding of ligand(growth-hormone) to formylated polymer gels, and the size exclusion column was connected in series to the affinity column. The crude growth-hormone receptor(Tritonx-100 extracts) obtained from rabbit liver homogenate was applied to the system. After elimination of unretained proteins, the receptor was eluted out from the affinity coulmn with 6-M urea solution. The eluent was led directly to the size exclusion column, where the receptor was readily desalted. the final eluent detected by uv absorption at 280 nm was fractionated, and the binding activity was assayed with use of <@1125<@d1i-labelled growth-hormone. as a result, from 16 mg of crude protein in the tritonx-100 extracts, 1200-fold purified receptor with a 10% recovery of binding activity was obtained within 4 hrs in a single application to the present system. The efficiency of the system was further enhaced by improving the method for preparation of affinity gels. The ligand was immobilized to aminated hard polymer gels by using alkali-treated glutaraldehyde(GA) as a cross-linker. The activity of the ligand was markedly higher than that obtained by using untreated GA which has been employed in the conventional method, while the amount of protein immobilized was almost unchanged. GA oligomers formed in the alkalitreated GA solution were responsible for this high activity
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