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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