4gla

Publication Abstract from PubMed

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. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naive combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naive OBody 11,700-fold over several affinity maturation steps, using phage display. 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. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. 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.

Tracking Molecular Recognition at the Atomic Level with a New Protein Scaffold Based on the OB-Fold.,Steemson JD, Baake M, Rakonjac J, Arcus VL, Liddament MT PLoS One. 2014 Jan 20;9(1):e86050. doi: 10.1371/journal.pone.0086050. eCollection, 2014 Jan 20. PMID:24465865^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.