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Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Engineering / Faculty of Engineering >
Peer-reviewed Journal Articles, etc >
金属キレートポリマーを導入したミセル水性二相分配法におけるヒスチジンタグ融合チトクロムb5及びオリゴペプチドのアフィニティー分配
| Title: | 金属キレートポリマーを導入したミセル水性二相分配法におけるヒスチジンタグ融合チトクロムb5及びオリゴペプチドのアフィニティー分配 |
| Other Titles: | Affinity partitioning of histidine-tagged cytochrome b5 and an oligopeptide in an aqueous micellar two-phase system containing a metal-chelating polymer |
| Authors: | 前花, 浩志1 Browse this author | | 谷, 博文2 Browse this author →KAKEN DB | | 鎌滝, 哲也3 Browse this author | | 上舘, 民夫4 Browse this author |
| Authors(alt): | Maehana, Koji1 | | Tani, Hirofumi2 | | Kamataki, Tetsuya3 | | Kamidate, Tamio4 |
| Keywords: | aqueous micellar two-phase system | | metal-chelating polymer | | affinity partitioning | | histidine-tagged membrane protein |
| Issue Date: | Jan-2002 |
| Publisher: | 日本分析化学会 |
| Journal Title: | BUNSEKI KAGAKU |
| Volume: | 51 |
| Issue: | 1 |
| Start Page: | 13 |
| End Page: | 19 |
| Publisher DOI: | 10.2116/bunsekikagaku.51.13 |
| Abstract: | A metal affinity interaction between metals and proteins was exploited for the selective partitioning of histidine-tagged membrane proteins in an aqueous micellar two-phase system. The system used was a mixture of non-ionic surfactant micelle, Triton X-100, and a metal-chelating polymer, Ni(II)-imminodiacetic acid-poly (ethylene glycol) (Ni-IDA-PEG). The mixture separated into two phases, a polymer-enriched upper phase and a micelle-enriched lower phase, under an appropriate condition. The effect of the Ni-IDA-PEG concentration on the partitioning of cytochrome b5 with and without a histidine-tag, and a fluorescein isothiocyanate-labeled oligopeptide containing six histidines was examined in two-phase systems containing various pH buffers. Cytochrome b5 without a histidine-tag was partitioned into a micelle-rich phase, independent of the Ni-IDA-PEG concentration, due to a hydrophobic interaction. On the other hand, with increasing the concentration of Ni-IDA-PEG, the partitioning of histidine-tagged cytochrome b5 was shifted to the polymer-rich phase in a system containing a phosphate or borate buffer. Upon the addition of imidazole to, or the removal of Ni(II) from the two-phase systems, the histidine-tagged protein showed the same partition behavior as that of the protein without a histidine-tag. These results indicate that the metal-chelating polymer plays the role of an affinity ligand, which enables histidine-tagged membrane proteins to move out from the micelle-rich phase to the polymer-rich phase. When Tris was used as a pH buffer, no affinity partitioning of the protein was observed. Although the oligopeptide also partitioned into the polymer phase in the presence of Ni-IDA-PEG, the partitioning was not dependent on the buffer type. Thus, the partitioning seems to be largely affected by the buffer type as well as the protein structure. The present system has a potential for a simple and rapid purification method for the histidine-tagged membrane proteins. | | 非イオン界面活性剤であるTriton X-100を用いたミセル水性二相分配系にNiIIを配位したイミノジ酢酸ポリエチレングリコール (Ni-IDA-PEG) を導入し, NiIIとのアフィニティー相互作用による膜タンパク質チトクロムb5及びオリゴペプチドの分配の制御を試みた. チトクロムb5は, Ni-IDA-PEG濃度に依存せずミセル相に分配したのに対し, ヒスチジンタグを融合したチトクロムb5及びオリゴペプチドはNi-IDA-PEG濃度の増加に伴いポリマー相に分配された. NiIIを配位していないIDA-PEG並びにイミダゾールを用いた実験から, ヒスチジンタグ融合チトクロムb5のポリマー相への分配は, ヒスチジンとNiIIとのアフィニティー相互作用によることが分かった. 更に, ヒスチジンタグ融合チトクロムb5のアフィニティー分配はpH緩衝剤の種類に大きく依存することが明らかとなった. |
| Type: | article |
| URI: | http://hdl.handle.net/2115/71715 |
| Appears in Collections: | 工学院・工学研究院 (Graduate School of Engineering / Faculty of Engineering) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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Submitter: 谷 博文
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