title: Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications.
abstract: We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.
authors.affiliation: Biomedical Health Research Centre, BioScreening Technology Group, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Tiede
authors.forename: Anna A S
authors.initials: AA
authors.lastname: Tang
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Deacon
authors.forename: Upasana
authors.initials: U
authors.lastname: Mandal
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Nettleship
authors.forename: Robin L
authors.initials: RL
authors.lastname: Owen
authors.forename: Suja E
authors.initials: SE
authors.lastname: George
authors.forename: David J
authors.initials: DJ
authors.lastname: Harrison
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
authors.forename: Darren C
authors.initials: DC
authors.lastname: Tomlinson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McPherson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D015891:Cystatins / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D025842:SUMO-1 Protein / Q000378:metabolism; D013696:Temperature
chemical_list: D015891:Cystatins; D019151:Peptide Library; D010455:Peptides; D025842:SUMO-1 Protein
keywords: SUMO; consensus protein; high specificity binding; non-antibody-binding protein; protein–protein interaction