Coupling protein design and in vitro selection strategies

Improving specificity and affinity of a designed β-protein IL-6 antagonist

Franck Martin, Carlo Toniatti, Anna Laura Salvati, Gennaro Ciliberto, Riccardo Cortese, Maurizio Sollazzo

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The minibody is a designed small β-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two β-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for 'stringent' selection such as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.

Original languageEnglish
Pages (from-to)86-97
Number of pages12
JournalJournal of Molecular Biology
Volume255
Issue number1
DOIs
Publication statusPublished - Jan 12 1996

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Bacteriophages
Interleukin-6
Epitopes
Cytokines
Proteins
Amino Acid Substitution
In Vitro Techniques
Immunoglobulin Domains

Keywords

  • Affinity selection
  • Hypervariable regions
  • Interleukin-6 antagonist
  • Phage display
  • Protein engineering

ASJC Scopus subject areas

  • Virology

Cite this

Coupling protein design and in vitro selection strategies : Improving specificity and affinity of a designed β-protein IL-6 antagonist. / Martin, Franck; Toniatti, Carlo; Salvati, Anna Laura; Ciliberto, Gennaro; Cortese, Riccardo; Sollazzo, Maurizio.

In: Journal of Molecular Biology, Vol. 255, No. 1, 12.01.1996, p. 86-97.

Research output: Contribution to journalArticle

Martin, Franck ; Toniatti, Carlo ; Salvati, Anna Laura ; Ciliberto, Gennaro ; Cortese, Riccardo ; Sollazzo, Maurizio. / Coupling protein design and in vitro selection strategies : Improving specificity and affinity of a designed β-protein IL-6 antagonist. In: Journal of Molecular Biology. 1996 ; Vol. 255, No. 1. pp. 86-97.
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