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Related Experiment Videos

Protein refolding at high concentrations using detergent/phospholipid mixtures

G Zardeneta1, P M Horowitz

  • 1Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760.

Analytical Biochemistry
|May 1, 1994
PubMed
Summary

This study demonstrates a novel method for refolding the sulfurtransferase enzyme rhodanese using mixed detergent-phospholipid micelles, achieving high yields of active protein. This technique enhances protein folding efficiency and aids in large-scale production of therapeutic proteins.

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Area of Science:

  • Biochemistry
  • Protein Chemistry
  • Biotechnology

Background:

  • Protein misfolding and aggregation are significant challenges in producing active therapeutic proteins.
  • Traditional refolding methods using detergents or chaperonins have limitations in yield and efficiency.
  • The sulfurtransferase enzyme rhodanese (EC 2.8.1.1) serves as a model for studying protein refolding.

Purpose of the Study:

  • To develop an efficient method for refolding rhodanese.
  • To investigate the role of mixed micellar structures in promoting correct protein folding.
  • To establish a scalable methodology for producing active, medically important proteins.

Main Methods:

  • Refolding of rhodanese was performed using mixtures of detergents (Triton X-100 or lauryl maltoside) and phospholipids.

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  • Varying protein concentrations (0.02 mg/ml and 0.2 mg/ml) were tested.
  • Comparison of yields with detergent-only and chaperonin-assisted refolding.

    • Main Results:

    • High yields (94%) of active rhodanese were achieved at lower concentrations (0.02 mg/ml) using Triton X-100/phosphatidylglycerol mixed micelles.
    • Significant yields (>= 45%) were obtained at higher concentrations (0.2 mg/ml) with detergent-phospholipid mixtures.
    • The mixed micelle system demonstrated superior refolding yields compared to detergents or chaperonins alone.

    Conclusions:

    • Mixed detergent-phospholipid micelles, forming large structures, effectively prevent misfolding and aggregation while promoting correct protein folding.
    • The amphipathic nature of mixed micelles facilitates binding to various sites on unfolded proteins and intermediates.
    • This methodology offers a promising approach for the large-scale production of active, biologically relevant proteins.