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Related Experiment Videos

Rapid method for measuring ScFv thermal stability by yeast surface display.

Brent A Orr1, Lori M Carr, K Dane Wittrup

  • 1Department of Biochemistry, University of Illinois, 600 S. Matthews Ave., Urbana, Illinois 61801, USA.

Biotechnology Progress
|April 5, 2003
PubMed
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We developed a simple yeast-surface method to measure single-chain antibody thermal stability. This technique is reproducible and helps compare protein structures with their stability for selecting antibody fragments.

Area of Science:

  • Biotechnology
  • Protein Engineering
  • Immunology

Background:

  • Assessing protein thermal stability is crucial for antibody engineering.
  • Existing methods for monitoring protein denaturation can be complex or time-consuming.

Purpose of the Study:

  • To develop and validate a simplified method for determining the relative thermal stability of single-chain variable fragments (scFv).
  • To enable rapid comparisons between protein structure and stability.

Main Methods:

  • Utilized flow cytometry to monitor irreversible denaturation of scFv fusions displayed on the yeast surface.
  • Quantitated thermal stability by measuring half-maximum denaturation temperatures.

Main Results:

  • The developed method demonstrated high reproducibility.

Related Experiment Videos

  • Results correlated well with established methods for monitoring soluble protein thermal denaturation.
  • A range of thermal stabilities (43–61°C) was observed for wild-type scFv antibodies.

    • Conclusions:

    • Immobilizing proteins on yeast surfaces allows for efficient quantitation of thermal stability.
    • This method facilitates rapid comparisons of primary structural information with protein stability.
    • Thermal denaturation is a valuable parameter for selecting scFv fragments for specific applications.