Kanamycin was added to 50 ug/mL and the culture incubated overnight at 250 rpm, 37C. (149K) GUID:?2999BB87-EAEE-43AC-8AC4-A96DDE2AE126 Figure S5: Sequence plan showing detailed construction method for the first affinity-maturation library. Blue arrows represent mutagenic oligonucleotides. Green arrows represent non-mutagenic oligonucleotides used for amplifying individual fragments and performing final gene reconstruction. Refer to Table S1 for oligonucleotide sequences.(TIF) pone.0086050.s005.tif (151K) GUID:?32A4198C-FF58-4472-AC78-1791C03D6755 Table S1: Oligonucleotide sequences. (XLSX) pone.0086050.s006.xlsx (12K) GUID:?E2777678-C5C3-4D71-B936-7B8F1D783990 Abstract 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 through protein engineering for specific molecular recognition. Antibodies and antibody fragments are currently the dominant class of engineered proteins for molecular recognition; very large combinatorial libraries (in excess of 1011 individual members) have been made and selected to obtain binding reagents with AURKA very high affinity and specificity (reviewed in [4]C[6]). However, some general limitations of the antibody format exist, such as their large size, requirement for glycosylation (in the case of full-length antibodies) and a critical dependence on intrachain disulfide bonds, a limitation shared by even the smallest fragments of antibodies UPF-648 (individual V domains, termed domain antibodies [7]). Driven by a desire to circumvent these limitations, a need to expand the useful target range that can be addressed pharmacologically and also to avoid potentially limiting issues of intellectual property, the field of non-antibody protein scaffolds has developed rapidly over UPF-648 the last decade (recently reviewed in [8]C[10]). Affibodies (based on UPF-648 a three-helix-bundle Z domain from staphylococcal protein A), have achieved high affinities against a range of protein targets [11]C[13]. Anticalins are high-affinity small-molecule UPF-648 binders that were selected from libraries of UPF-648 a mutant lipocalin fold [14]. Designed Ankyrin repeat proteins (DARPins) have been engineered with sub-nanomolar affinities for protein targets [15]. The Sac7d DNA-binding protein, an SH3-like five-stranded incomplete -barrel (mistakenly identified as an OB-fold [16]) from the hyperthermophile has successfully been used as a scaffold to produce high-affinity binders to a range of targets C these were initially called Affitins, but later renamed Nanofitins [17]. Engineered derivatives of variable lymphocyte receptors, termed Repebodies, have also recently been described with affinities in the nanomolar range [18]. Given the large number of proteins investigated for engineering purposes, there are relatively few crystal structures of engineered protein-protein complexes. Hogbom et al [19] claimed the first example with an Affibody, and other examples have been published, most notably those of an Anticalin [20], a DARPin [21] and two Repebodies [18]. In principle, OB-folds possess features granting potential benefits to an engineered OB-fold domain over and above antibodies based on the immunoglobulin fold; they are small, single-domain binding modules and generally lack disulphide bonds. Due to their ubiquitous nature, potential OB-fold-based scaffolds may be sourced from widely diverse organisms, including human proteins for therapeutic and diagnostic use, or thermophilic microorganisms for more general applications. Given that OB-folds have a unique antigen binding site (formed from a combination of concave -sheet and loops), it may be possible to favour different binding solutions than those seen in scaffolds where loops are the principal mediators of specificity and.