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 credited.įunding: This project was funded by a grant from the Marsden Fund of New Zealand with additional funds from the Maurice Wilkins Centre for Molecular Biodiscovery, New Enterprise Research Fund, AgResearch Limited, The University of Waikato and OBodies Ltd. Received: SeptemAccepted: DecemPublished: January 20, 2014Ĭopyright: © 2014 Steemson et al. PLoS ONE 9(1):Įditor: Stefan Dübel, Technical University of Braunschweig, Germany These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.Ĭitation: Steemson JD, Baake M, Rakonjac J, Arcus VL, Liddament MT (2014) Tracking Molecular Recognition at the Atomic Level with a New Protein Scaffold Based on the OB-Fold. They can be expressed in soluble form and also purified from bacteria at high yields. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. For this single-domain scaffold we have coined the term OBody. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands.
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