Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 27, Issue 5, Pages 145-155Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzu007
Keywords
consensus protein; high specificity binding; non-antibody-binding protein; protein-protein interaction; SUMO
Funding
- Biotechnology and Biological Sciences Research Council [24/G15882]
- University of Leeds Transformation Fund
- WELMEC, a Centre of Excellence in Medical Engineering - Wellcome Trust
- EPSRC [WT 088908/Z/09/Z]
- MRC
- BBSRC
- MRC [MR/K018779/1] Funding Source: UKRI
- Medical Research Council [MR/K018779/1] Funding Source: researchfish
Ask authors/readers for more resources
We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (T-m ca. 101 degrees C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 angstrom 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 x 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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available