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 language | English |
|---|---|
| Pages (from-to) | 86-97 |
| Number of pages | 12 |
| Journal | Journal of Molecular Biology |
| Volume | 255 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Jan 12 1996 |
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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 journal › Article
}
TY - JOUR
T1 - Coupling protein design and in vitro selection strategies
T2 - Improving specificity and affinity of a designed β-protein IL-6 antagonist
AU - Martin, Franck
AU - Toniatti, Carlo
AU - Salvati, Anna Laura
AU - Ciliberto, Gennaro
AU - Cortese, Riccardo
AU - Sollazzo, Maurizio
PY - 1996/1/12
Y1 - 1996/1/12
N2 - 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.
AB - 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.
KW - Affinity selection
KW - Hypervariable regions
KW - Interleukin-6 antagonist
KW - Phage display
KW - Protein engineering
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U2 - 10.1006/jmbi.1996.0008
DO - 10.1006/jmbi.1996.0008
M3 - Article
VL - 255
SP - 86
EP - 97
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 1
ER -