Skip to main content
Article thumbnail
Location of Repository

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

By Andreas Hoffmann, Michael Kovermann, Hauke Lilie, Markus Fiedler, Jochen Balbach, Rainer Rudolph and Sven Pfeifer


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

Topics: Research Article
Publisher: Public Library of Science
OAI identifier:
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles


    1. (2007). A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2.
    2. (2011). a Subunit Fracture Mechanism.
    3. (2007). Affilin-Novel Binding Molecules Based on Human [gamma]-B-Crystallin, an All [beta]-Sheet Protein.
    4. (1998). Anion binding to the ubiquitin molecule.
    5. (1992). Antigenic regions of tumor necrosis factor alpha and their topographic relationships with structural/ functional domains.
    6. (2005). Artificial, non-antibody binding proteins for pharmaceutical and industrial applications. Trends Biotechnol 23: 514–522. New Binding Mode to TNF-Alpha PLoS
    7. (1994). Assignment of the Contribution of the Tryptophan Residues to the Circular Dichroism Spectrum of Human Carbonic Anhydrase II.
    8. (2002). Binding and Functional Comparisons of Two Types of Tumor Necrosis Factor Antagonists.
    9. (2006). Binding Characteristics of Tumor Necrosis Factor Receptor-Fc Fusion Proteins vs Anti-Tumor Necrosis Factor mAbs.
    10. (1997). Binding proteins selected from combinatorial libraries of an [alpha]-helical bacterial receptor domain.
    11. (1999). Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.
    12. (1993). Circular dichroism studies of barnase and its mutants: Characterization of the contribution of aromatic side chains.
    13. (1995). Comparison of Reactivity of Monoclonal Antibody (3F2) to Trimeric Tumor Necrosis Factor (TNF-alpha) with That to Monomeric TNF-alpha.
    14. (2009). Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor.
    15. (1993). Corti A
    16. (2007). Efficient protein selection based on ribosome display system with purified components.
    17. (1992). Fast internal main-chain dynamics of human ubiquitin.
    18. (1993). Folding and stability of a tryptophan-containing mutant of ubiquitin.
    19. (2004). Generation of artificial binding proteins on the basis of ubiquitin proteins. International Patent Application WO/2004/
    20. (2009). Generation of tumour-necrosis-factor - specific affibody molecules capable of blocking receptor binding in vitro.
    21. (1994). Guiding the Selection of Human Antibodies from Phage Display Repertoires to a Single Epitope of an Antigen.
    22. (1995). Homologous ELISA for detection of oligomeric human TNF: properties of the assay.
    23. (1995). Improved Sensitivity of HSQC Spectra of Exchanging Protons at Short Interscan Delays Using a New Fast HSQC (FHSQC) Detection Scheme That Avoids Water Saturation.
    24. (1995). Mechanism of suramin-induced deoligomerization of tumor necrosis factor.
    25. (2002). Molecular recognition in antibody-antigen complexes.
    26. (2009). NMR studies on binding sites and aggregation-disassociation of fluorinated surfactant sodium perfluorooctanoate on protein ubiquitin.
    27. (1994). NMR View: A computer program for the visualization and analysis of NMR data.
    28. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes.
    29. (2009). Nonproteolytic Functions of Ubiquitin in Cell Signaling.
    30. (1997). Optical spectroscopy to characterize protein conformation and conformational changes.
    31. (2003). Plu ¨ckthun A
    32. (2007). Pluckthun A
    33. (1990). Plueckthun A
    34. (2005). Protein production by auto-induction in high-density shaking cultures.
    35. (2010). Recombinant production of bioactive human TNF-[alpha] by SUMO-fusion system - High yields from shake-flask culture.
    36. (2010). Selection of a Novel and Highly Specific Tumor Necrosis Factor [alpha] (TNF [alpha
    37. (2009). Skerra A
    38. (2005). Smallmolecule inhibition of TNF-{alpha}.
    39. (2003). Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding.
    40. (2010). Structural and mechanistic insight into how antibodies inhibit serine proteases.
    41. (2003). Structure and Ubiquitin Binding of the Ubiquitin-interacting Motif.
    42. (1994). Structure-activity studies of human tumour necrosis factors.
    43. (1987). The active form of tumor necrosis factor is a trimer.
    44. (2006). Ubiquitin-binding domains.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.