Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications
| Autor: | Christian, Tiede, Anna A S, Tang, Sarah E, Deacon, Upasana, Mandal, Joanne E, Nettleship, Robin L, Owen, Suja E, George, David J, Harrison, Raymond J, Owens, Darren C, Tomlinson, Michael J, McPherson |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Base Sequence Protein Stability consensus protein Molecular Sequence Data SUMO-1 Protein Temperature Original Articles Protein Engineering Cystatins high specificity binding Protein Structure Secondary non-antibody-binding protein protein–protein interaction Peptide Library SUMO Consensus Sequence Humans Amino Acid Sequence Peptides |
| Zdroj: | Protein Engineering, Design and Selection |
| ISSN: | 1741-0134 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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