Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications

• Published in: Protein engineering, design and selection Vol. 27; no. 5; pp. 145 - 155
• Main Authors: Tiede, Christian, Tang, Anna A. S., Deacon, Sarah E., Mandal, Upasana, Nettleship, Joanne E., Owen, Robin L., George, Suja E., Harrison, David J., Owens, Raymond J., Tomlinson, Darren C., McPherson, Michael J.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.05.2014
• Subjects: Amino Acid Sequence; Base Sequence; Consensus Sequence; Cystatins - chemistry; Escherichia coli; Humans; Models, Molecular; Molecular Sequence Data; Original; Peptide Library; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Protein Engineering - methods; Protein Stability; Protein Structure, Secondary; SUMO-1 Protein - metabolism; Temperature; protein–protein interaction; SUMO; high specificity binding; non-antibody-binding protein; consensus protein
• ISSN: 1741-0126; 1741-0134; 1741-0134
• DOI: 10.1093/protein/gzu007

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 × 1010 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.