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Fine affinity discrimination by yeast surface display and flow cytometry.

J J VanAntwerp1, K D Wittrup

  • 1Department of Chemical Engineering, University of Illinois, 600 South Mathews, Urbana, Illinois 61801, USA.

Biotechnology Progress
|February 9, 2000
PubMed
Summary

Yeast surface display effectively enriches rare antibody variants. This protein engineering system achieves high purity and yield, crucial for antibody affinity maturation.

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Area of Science:

  • Protein engineering
  • Molecular biology
  • Biotechnology

Background:

  • Yeast surface display is a eukaryotic system for directed protein evolution.
  • Antibody affinity maturation requires high enrichment factors for rare clones.

Purpose of the Study:

  • To validate the purification potential of yeast surface display for antibody engineering.
  • To assess enrichment factors and purity for differentiating antibody variants with subtle affinity differences.

Main Methods:

  • Engineered Saccharomyces cerevisiae to display D1.3 antibody and a higher affinity mutant (M3).
  • Mixed M3-displaying cells with D1.3-displaying cells at a 1:1000 ratio.
  • Used flow cytometry for fluorescent labeling and cell sorting based on antigen binding.

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Main Results:

  • Achieved a single-pass enrichment factor of 125-fold (+/- 65-fold) for the M3 mutant.
  • Demonstrated precise and reproducible sorting under optimal labeling conditions.
  • Validated yeast display's capability for fine discrimination between mutant clones of similar affinity.

Conclusions:

  • Yeast surface display is a powerful tool for antibody affinity maturation due to its high enrichment and purity capabilities.
  • The system's ability to detect subtle affinity improvements is essential for rapid protein engineering and drug discovery.