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A synthetic mimic of a discontinuous binding site on interleukin-10

Nature Biotechnology volume 17, pages 271–275 (1999)Cite this article

Abstract

We synthetically reconstructed a discontinuous binding site on interleukin-10 (IL-10) that recognizes the neutralizing anti–IL-10 antibody CB/RS/1. To design the 32-mer IL-10 mimic, a discontinuous interaction site on IL-10 was mapped, and binding studies with epitope-derived peptides led to specific replacement of several amino acids. Both parts of the interaction site were combined by addition of a linker molecule. Systematic analoging of the combined molecule then led to introduction of several additional substitutions in both regions and the linker. All possible disulfide bridge–containing variants of the 32-mer were tested by binding studies. Parallel syntheses were performed on continuous cellulose membranes by spot synthesis. As a result, a conformationally stabilized IL-10–derived molecule was obtained that both binds to and neutralizes the biological activity of CB/RS/1 in the low nanomolar range. This synthetic approach is a powerful alternative to phage display methods for the design of protein mimics.

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Figure 1: Steps involved in the identification of an IL-10 mimic.
Figure 2: Competition for CB/RS/1 binding between solid phase-bound hIL-10 and pepIII, pepIV, pepV, and pepVI (Fig. 1) or free hIL-10.
Figure 3: Effect of pepVI on the neutralizing activity of CB/RS/1.

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Acknowledgements

We thank Christiane Landgraf and Marén Schlief for excellent technical assistance in peptide synthesis. This work was supported by grants of the Deutsche Forschungsgemeinschaft, Fonds der Chemischen Industrie, and the Charité.

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Author notes

  1. Ulrich Reineke

    Present address: JERINI BioTools GmbH, Ost-West-Zentrum, Rudower Chaussee 5, D-12489, Berlin

  2. Ulrich Reineke and Robert Sabat: Both authors contributed equally to this work.

Authors and Affiliations

  1. Institut für Medizinische Immunologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Schumannstr. 20/21, Berlin, D-10098

    Ulrich Reineke, Robert Sabat, Hans-Dieter Volk & Jens Schneider-Mergener

  2. Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Schumannstr. 20/21, Berlin, D-10098

    Rolf Misselwitz

  3. Max Delbrück Centrum für Molekulare Medizin , Robert-Rössle-Straβe 10, Berlin, D-13122, Germany

    Heinz Welfle

Authors
  1. Ulrich Reineke
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  6. Jens Schneider-Mergener
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Correspondence to Robert Sabat or Jens Schneider-Mergener.

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Reineke, U., Sabat, R., Misselwitz, R. et al. A synthetic mimic of a discontinuous binding site on interleukin-10. Nat Biotechnol 17, 271–275 (1999). https://doi.org/10.1038/7018

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