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Related Experiment Videos

Enhanced protein renaturation by temperature-responsive polymers.

S C Lin1, K L Lin, H C Chiu

  • 1Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan, Republic of China. sclin@dragon.nchu.edu.tw

Biotechnology and Bioengineering
|January 29, 2000
PubMed
Summary

Poly(N-isopropylacrylamide) (PNIPAAm) effectively renatures beta-lactamase from inclusion bodies, improving yield by 41%. This temperature-sensitive polymer offers enhanced protein renaturation efficiency and easy recycling.

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Area of Science:

  • Biochemistry
  • Polymer Science
  • Protein Engineering

Background:

  • Protein misfolding and aggregation into inclusion bodies is a common challenge in recombinant protein production.
  • Efficient renaturation of proteins from inclusion bodies is crucial for obtaining active biological molecules.
  • Polymeric additives like polyethylene glycol (PEG) have been used to enhance protein renaturation.

Purpose of the Study:

  • To investigate the efficacy of poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, in renaturing beta-lactamase from inclusion bodies.
  • To compare the performance of PNIPAAm with PEG in enhancing protein renaturation.
  • To explore the mechanism and optimal conditions for polymer-assisted protein renaturation.

Main Methods:

  • Inclusion bodies of beta-lactamase were prepared and subjected to renaturation protocols.

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  • The effect of PNIPAAm and PEG on renaturation yield and kinetics was evaluated at various concentrations and temperatures.
  • A sequential addition approach was employed to reduce local protein concentration during renaturation.
  • Kinetic studies were performed to analyze the impact of polymers on the initial rate and final yield of protein activity.

    • Main Results:

    • PNIPAAm significantly enhanced beta-lactamase activity yield by 41% (to 65.4 IU/mL) at 0.1% concentration, outperforming PEG (26% increase to 58.7 IU/mL).
    • PNIPAAm increased the final activity yield without significantly affecting the initial renaturation rate.
    • Hydrophobic interactions between denatured proteins and polymers were identified as a key factor in enhanced renaturation, with activity increasing with temperature.
    • The sequential addition method, particularly with PNIPAAm, further boosted activity yield by 60.5% (to 74.6 IU/mL).

    Conclusions:

    • PNIPAAm is a highly effective polymer for enhancing the renaturation of beta-lactamase from inclusion bodies.
    • The mechanism of enhancement by PNIPAAm and PEG appears similar, involving hydrophobic interactions.
    • PNIPAAm's temperature-sensitive nature allows for efficient separation and recycling, making it a promising tool for industrial protein refolding processes.
    • A proposed protein renaturation process utilizing PNIPAAm offers advantages in efficiency, minimal aggregate loss, and polymer recyclability.