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Related Experiment Videos

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

R Kuboi1, S Morita, H Ota

  • 1Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Japan. kuboi@cheng.es.osaka-u.ac.jp

Journal of Chromatography. B, Biomedical Sciences and Applications
|August 15, 2000
PubMed
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Stimuli-responsive polymers enhance protein refolding and separation in aqueous two-phase systems (ATPS). A novel poly(ethylene glycol) (PEG) ligand effectively suppressed aggregation and improved refolding yields for carbonic anhydrase from bovine (CAB).

Area of Science:

  • Biochemistry
  • Polymer Science
  • Separation Science

Background:

  • Protein misfolding and aggregation are significant challenges in biotechnology.
  • Aqueous two-phase systems (ATPS) offer a promising platform for protein separation and purification.
  • Stimuli-responsive polymers can be utilized to control protein behavior in solution.

Purpose of the Study:

  • To investigate the function of a stimuli-responsive polymer for protein unfolding and refolding in ATPS.
  • To enhance protein separation and purification using modified ATPS.
  • To explore the refolding of carbonic anhydrase from bovine (CAB) using a thermo-reactive ligand.

Main Methods:

  • Modification of the poly(ethylene glycol) (PEG) phase in ATPS with a thermo-reactive hydrophobic ligand (poly(propylene oxide)-phenyl group (PPO-Ph group)).

Related Experiment Videos

  • Examination of CAB refolding in the presence of PPO-Ph-PEG at various temperatures.
  • Characterization of local hydrophobicity changes using aqueous two-phase partitioning and a hydrophobic fluorescent probe.

    • Main Results:

    • Addition of PPO-Ph-PEG significantly enhanced CAB refolding yield and suppressed aggregate formation at 50-55°C.
    • The local hydrophobicity of CAB peaked at 60°C, while PPO-Ph-PEG hydrophobicity increased above 45°C.
    • A model for CAB refolding involving PPO-Ph-PEG complex formation and subsequent release of native CAB was proposed.

    Conclusions:

    • Stimuli-responsive polymers, specifically PPO-Ph-PEG, are effective in promoting protein refolding and preventing aggregation in ATPS.
    • Temperature-dependent changes in hydrophobicity play a crucial role in the mechanism of polymer-assisted protein refolding.
    • This approach offers a novel strategy for efficient protein separation and purification in biotechnological applications.