New binding mode to TNF-alpha revealed by ubiquitin-based artificial binding protein.

PloS One
Andreas HoffmannSven Pfeifer

Abstract

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 sc...Continue Reading

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Citations

May 2, 2015·Trends in Biotechnology·Katja ŠkrlecAleš Berlec
Jan 24, 2015·Protein Engineering, Design & Selection : PEDS·Stefan LuziPhilipp Holliger
Mar 1, 2019·The Journal of Biological Chemistry·Helena AndradeYixin Zhang
Jan 30, 2014·The Journal of Biological Chemistry·Susan LoreyUlrich Haupts
Jun 6, 2017·Bioengineering & Translational Medicine·Maryna Gorelik, Sachdev S Sidhu
Jul 15, 2020·ACS Chemical Biology·James W CheccoSamuel H Gellman

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Methods Mentioned

BETA
isothermal
NMR
chemical modification
ELISA
size exclusion chromatography
size
circular dichroism
size-exclusion chromatography
isothermal titration calorimetry
equilibrium sedimentation

Software Mentioned

NMRPipe
NMRView
PyMOL

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