研究者詳細 - 島内　寿徳

論文 - 島内　寿徳

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Stimuli-responsive separation of proteins using immobilized liposome chromatography

Toshinori Shimanouchi, Seiichi Morita, Hiroshi Umakoshi, Ryoichi Kuboi

Journal of Chromatography B: Biomedical Sciences and Applications 743 ( 1-2 ) 85 - 91 2000年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s, max) = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, k(s, max) = 11.3 at pH 4
carbonic anhydrase from bovine, k(s, max) = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the k(s)-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter. © 2000 Elsevier Science B.V.

DOI： 10.1016/S0378-4347(00)00061-X

Stimuli-responsive separation of proteins using immobilized liposome chromatography 査読

T Shimanouchi, S Morita, H Umakoshi, R Kuboi

JOURNAL OF CHROMATOGRAPHY B 743 ( 1-2 ) 85 - 91 2000年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of beta-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s,max) = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (alpha-glucosidase, k(s,max) = 11.3 at pH 4; carbonic anhydrase from bovine, k(s,max) = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the k(s)-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter. (C) 2000 Elsevier Science B.V. All rights reserved.

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Immobilized liposome chromatography for refolding and purification of protein 査読

M Yoshimoto, T Shimanouchi, H Umakoshi, R Kuboi

JOURNAL OF CHROMATOGRAPHY B 743 ( 1-2 ) 93 - 99 2000年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield. (C) 2000 Elsevier Science B.V. All rights reserved.

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Selective separation process of proteins based on the heat stress- induced translocation across phospholipid membranes

Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

Journal of Chromatography B: Biomedical Applications 711 ( 1-2 ) 111 - 116 1998年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Heating of several protein solutions at 40-47°C for 5-60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of β-galactosidase (β-gal), α-glucosidase (α- glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of β-gal at various temperatures were maximized under suitable heating conditions (45°C, 30 min). Those of α-glu and CAB were maximized at 40-45 and 60°C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of β-gal, α-glu and CAB.

DOI： 10.1016/S0378-4347(97)00659-2

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes 査読

H Umakoshi, T Shimanouchi, R Kuboi

JOURNAL OF CHROMATOGRAPHY B 711 ( 1-2 ) 111 - 116 1998年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Heating of several protein solutions at 40-47 degrees C for 5-60 min in the presence of 1-palmitoyl-2-oleoyI-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of beta-galactosidase (beta-gal), alpha-glucosidase (alpha-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of P-gal at various temperatures were maximized under suitable heating conditions (45 degrees C, 30 min). Those of alpha-glu and CAB were maximized at 40-45 and 60 degrees C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of P-gal, a-glu and CAB. (C) 1998 Elsevier Science B.V. All rights reserved.

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Model system for heat-induced translocation of cytoplasmic β-galactosidase across phospholipid bilayer membrane

Hiroshi Umakoshi, Makoto Yoshimoto, Toshinori Shimanouchi, Ryoichi Kuboi, Isao Komasawa

Biotechnology Progress 14 ( 2 ) 218 - 226 1998年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) in which β-galactosidase (β-gal) was entrapped. Exposing the POPC liposomes entrapping β-gal inside to heat treatment (40-50°C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of β-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping β-gal were heated at 45°C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45°C, 30 min) also supported the translocation of β-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of β-gal was maximized at the temperature of 40-50 °C. The mechanisms of β-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and β-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of β-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of β-gal were well corresponding to those from model calculation.

DOI： 10.1021/bp9800135

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