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Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems.

R Kuboi·S Morita·H Ota·H Umakoshi
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Abstract

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50-55 degrees C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60 degrees C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45 degrees C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.

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AnimalsCarbonic AnhydrasesCattleHot TemperaturePolyethylene GlycolsProtein FoldingTemperatureWaterJournal ArticleResearch Support, Non-U.S. Gov't

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R Kuboi, S Morita, H Ota, H Umakoshi. 2000-06-23. Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems.. https://doi.org/10.1016/s0378-4347(00)00062-1

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