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Yeast Surface Display and Cell Sorting of Antigen-Binding Fc Fragments

  • Flávio Sádio
  • Gerhard Stadlmayr
  • Katharina Stadlbauer
  • Florian Rüker
  • Gordana Wozniak-KnoppEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)

Abstract

Since the introduction of the yeast display platform, this method has increasingly gained popularity for the discovery and affinity maturation of antibodies and other protein scaffolds intended for antigen recognition. Yeast display is particularly well suited for the selection of antigen-binding Fc fragments (Fcabs) as it allows rapid combinatorial library construction via gap repair-driven homologous recombination and an efficient display of a glycosylated Fc able to interact with Fcγ receptors. Apart from expression-related normalization, isolation of properly folded Fcabs can be guided efficiently by simultaneous staining with ligands such as protein A, FcγRI, or the conformation-sensitive anti-FigCH2 antibody, whose binding is critically dependent on the integrity of the Fc structure. The particular properties of the Fcab scaffold, such as its homodimeric state which can promote binding to multiple antigen molecules, require modifications of traditional affinity maturation strategies. Preferred to equilibrium selections are kinetically driven antigen selections, designed to specifically influence the binding off-rate, which in many cases augments the desired biological effect. A simple design of a yeast-displayed heterodimeric Fc fragment is described and can be used as a general guideline for affinity selection of Fcabs with an asymmetric binding site. Overall, this chapter underlines the importance of the versatile yeast display technique for the optimization of the novel Fcab scaffold for antigen recognition.

Key words

Fcab Yeast display Affinity maturation Directed evolution Heterodimer 

Notes

Acknowledgments

The financial support by the Austrian Federal Ministry for Digital and Economic Affairs and the National Foundation for Research, Technology and Development is gratefully acknowledged. The company F-star has supported this work as well.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Flávio Sádio
    • 1
  • Gerhard Stadlmayr
    • 1
  • Katharina Stadlbauer
    • 1
  • Florian Rüker
    • 1
  • Gordana Wozniak-Knopp
    • 1
    Email author
  1. 1.Christian Doppler Laboratory for Innovative Immunotherapeutics, Department of BiotechnologyUniversity of Natural Resources and Life Sciences (BOKU)ViennaAustria

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