Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems.
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.
Kuboi
R
R
Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Japan. kuboi@cheng.es.osaka-u.ac.jp
Morita
S
S
Ota
H
H
Umakoshi
H
H
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D006358:Hot Temperature; D011092:Polyethylene Glycols / Q000737:chemistry*; D017510:Protein Folding; D013696:Temperature; D014867:Water / Q000737:chemistry
D014867:Water; D011092:Polyethylene Glycols; D002256:Carbonic Anhydrases