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Related Experiment Videos

Directed evolution for the development of conformation-specific affinity reagents using yeast display.

Jane M Weaver-Feldhaus1, Keith D Miller, Michael J Feldhaus

  • 1Pacific Northwest National Laboratory, Richland, WA 99352, USA.

Protein Engineering, Design & Selection : PEDS
|September 28, 2005
PubMed
Summary

Researchers used yeast display to create conformation-specific antibodies for calmodulin (CaM). This directed evolution method rapidly generates high-affinity antibodies targeting specific CaM states, useful for proteomic applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunology

Background:

  • Yeast display is a key technique for antibody engineering, enhancing affinity and stability.
  • Mammalian calmodulin (CaM) is a crucial signaling protein with distinct structural conformations upon calcium binding.
  • Conformation-specific antibodies are valuable tools for proteomic research.

Purpose of the Study:

  • To develop conformation-specific single-chain variable fragment (scFv) antibodies against distinct states of mammalian calmodulin (CaM).
  • To utilize yeast display and directed evolution for rapid antibody generation and optimization.
  • To establish flow cytometry-based screening for isolating antibodies targeting Ca(2+)-bound CaM (Ca(2+)-CaM) and Ca(2+)-free CaM (apo-CaM).

Main Methods:

  • Employed yeast display technology for antibody library construction and screening.

Related Experiment Videos

  • Utilized flow cytometry for high-throughput screening of scFv clones against CaM conformational states.
  • Applied directed evolution to enhance antibody affinity and specificity for apo-CaM.

    • Main Results:

    • Isolated high-affinity (K(d) = 0.8 nM) and specific (>1000-fold) scFv antibodies targeting Ca(2+)-CaM.
    • Developed antibodies with improved affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM through directed evolution.
    • Observed an unexpected loss of CaM-binding activity upon conversion of selected clones into soluble fragments.

    Conclusions:

    • Yeast display facilitates the rapid isolation of conformation-specific antibodies against CaM.
    • Directed evolution is effective for optimizing antibody affinity and specificity for target protein isoforms.
    • The study demonstrates a robust platform for generating custom antibodies for proteomic studies.