Design of Optimum Refolding Solution by Combination of Reagents Classified by Specific Function
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概要
- 論文の詳細を見る
The effect of guanidinium chloride on protein unfolding was regarded as a combination of the effects on interactions such hydrophobic interaction or hydrogen bond, and ionic interaction that construct a higher-order structure of the protein. Urea and LiCl instead of guanidinium chloride were used for refolding of denatured reduced lysozyme by assuming that urea and LiCl individually control hydrophobic interaction or hydrogen bond and ionic interaction, respectively. The refolding media were prepared using various combinations of urea and LiCl concentrations. Reoxidation of thiol groups was carried out in a glutathione redox system. The recovered enzyme activity at a lysozyme concentration of 10 μM was usually less than 20% in 50 mM Tris buffer due to irreversible misfolding and/or aggregation during refolding. In the presence of urea and LiCl at high concentrations the recovered activity increased markedly. Complete renaturation could be achieved in a broad range of concentrations of urea and LiCl around the optimum of 3 M urea and 1.5 M LiCl, while it could not be achieved solely with guanidinium chloride. The availability of the concept that multifunction of guanidinium chloride can be separated approximately into two monofunctions of urea and LiCl was confirmed by the optimum design of refolding media prepared by combinations of urea and LiCl. A characteristic diagram for the refolding of lysozyme was depicted. This diagram will aid the optimum design of refolding media and study of the refolding mechanism.
- 社団法人日本生物工学会の論文
- 1996-10-25
著者
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野原 大輔
名古屋市立大学薬学部 薬品製造工学教室
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酒井 朝也
名古屋市立大学薬学部 薬品製造工学教室
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NOHARA DAISUKE
Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City Univers
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MATSUBARA MAMORU
Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City Univers
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SAKAI TOMOYA
Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City Univers
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TANO KOUJI
Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City Univers
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Tano Kouji
Department Of Chemical Reaction Engineering Faculty Of Pharmaceutical Sciences Nagoya City Universit
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Sakai Tomoya
Department Of Chemical Reaction Engineering Faculty Of Pharmaceutical Sciences Nagoya City Universit
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Nohara Daisuke
Department Of Biomolecular Science Faculty Of Engineering Gifu University
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Matsubara Mamoru
Department Of Chemical Reaction Engineering Faculty Of Pharmaceutical Sciences Nagoya City Universit
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