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New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target...

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Autores principales: Hoffmann, Andreas, Kovermann, Michael, Lilie, Hauke, Fiedler, Markus, Balbach, Jochen, Rudolph, Rainer, Pfeifer, Sven
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282696/
https://www.ncbi.nlm.nih.gov/pubmed/22363609
http://dx.doi.org/10.1371/journal.pone.0031298
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author Hoffmann, Andreas
Kovermann, Michael
Lilie, Hauke
Fiedler, Markus
Balbach, Jochen
Rudolph, Rainer
Pfeifer, Sven
author_facet Hoffmann, Andreas
Kovermann, Michael
Lilie, Hauke
Fiedler, Markus
Balbach, Jochen
Rudolph, Rainer
Pfeifer, Sven
author_sort Hoffmann, Andreas
collection PubMed
description A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.
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spelling pubmed-32826962012-02-23 New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein Hoffmann, Andreas Kovermann, Michael Lilie, Hauke Fiedler, Markus Balbach, Jochen Rudolph, Rainer Pfeifer, Sven PLoS One Research Article A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. Public Library of Science 2012-02-20 /pmc/articles/PMC3282696/ /pubmed/22363609 http://dx.doi.org/10.1371/journal.pone.0031298 Text en Hoffmann et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Hoffmann, Andreas
Kovermann, Michael
Lilie, Hauke
Fiedler, Markus
Balbach, Jochen
Rudolph, Rainer
Pfeifer, Sven
New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
title New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
title_full New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
title_fullStr New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
title_full_unstemmed New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
title_short New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
title_sort new binding mode to tnf-alpha revealed by ubiquitin-based artificial binding protein
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282696/
https://www.ncbi.nlm.nih.gov/pubmed/22363609
http://dx.doi.org/10.1371/journal.pone.0031298
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