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

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Book cover Recombinant Protein Production in Yeast

Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  1. Christian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

    Flávio Sádio, Gerhard Stadlmayr, Katharina Stadlbauer, Florian Rüker & Gordana Wozniak-Knopp

Authors
  1. Flávio Sádio
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  2. Gerhard Stadlmayr
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  3. Katharina Stadlbauer
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  4. Florian Rüker
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  5. Gordana Wozniak-Knopp
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Corresponding author

Correspondence to Gordana Wozniak-Knopp .

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Editors and Affiliations

  1. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Brigitte Gasser

  2. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Diethard Mattanovich

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Sádio, F., Stadlmayr, G., Stadlbauer, K., Rüker, F., Wozniak-Knopp, G. (2019). Yeast Surface Display and Cell Sorting of Antigen-Binding Fc Fragments. In: Gasser, B., Mattanovich, D. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 1923. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9024-5_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9024-5_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9023-8

  • Online ISBN: 978-1-4939-9024-5

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