Dr Darren Tomlinson
Bioscreening Technology Group Manager
School of Molecular and Cellular Biology
Contact: Astbury 7.108 and Level 8 WTBB - LIMM | +44(0) 113 34 37099 and 34 38445 |
Antibodies are the best-studied group of biological binding molecules to date. They are important in a wide variety of biological and medical applications, but as molecular biology reagents they are limited by their significant size, poor stability, production costs and batch-to-batch variation. To overcome these issues a number of alternative binding reagents (protein, RNA and DNA aptamers) have been developed. These can bind to epitopes on target proteins and so have potential as molecular biology tools, therapeutic agents and as diagnostic tools for detection and imaging of proteins in patient samples. Our group was established to exploit a novel artificial binding protein (ABP) library. Our ABP is called an adhiron and is based on a constant small 91 amino acid scaffold protein that constrains two randomised nine amino acid loop regions for molecular recognition (Figure 1A). The scaffold protein is extremely stable with a Tm of 101oC and is the most stable ABP scaffold to date (Figure 1B), and maintains the beta structure following loop insertion (Figure 1C). We have developed a large naïve phage display library (>3x1010) of adhirons that is of very high quality (86 % full length clones). The loop regions in the library contain an even distribution of each of the 19 amino acids excluding cysteines (Figure 1D). We work collaboratively with academics and clinicians on numerous projects. A few examples are listed below
Inhibiting protein-protein interactions
We are working with Professor Adrian Whitehouse and Dr David Hughes to generate specific reagents to block human SUMO2 (hSUMO2), and for the first time we have developed ABP reagents which differentiate between hSUMO1 and hSUMO2 isoforms (Fig 2A). To confirm the ability to inhibit hSUMO2 binding they developed assays that test ABPs ability to inhibit SUMO interactions. In vitro recombinant RNF4 ubiquitinates polymers of hSUMO2 (poly-hSUMO22-8). ABPs specific for GFP (irrelevant control) and for hSUMO1 were unable to inhibit RNF4s ability to ubiquitinate poly-hSUMO22-8, whereas ABPs specific for hSUMO2 robustly inhibited this activity at less than 1 μM (Fig 2B).
Inhibiting protein function
We are working with Dr Ramzi Aijan to modulate blood clot formation or facilitate lysis (clot breakdown). We raised numerous adhirons against fibrinogen and used assays to assess changes in fibrin clot formation and lysis. Many adhirons either prolonged clot formation or altered lysis (Fig 3). Some binders completely inhibited lysis and others inhibited specific protein protein interactions. We aim to study fibrin clot formation and identify novel methods for modulating clotting in patients.
Identifying druggable domains
We are working with Prof Ann Morgan and Dr James Robinson who are validating a receptor as a therapeutic target in rheumatoid arthritis, and we have identified adhirons that block receptor function (TNF release and phagocytosis). Three of the adhiron receptor complexes have been co-crystallised in collaboration with Dr Jo Nettleship and Dr Ray Owens at the Oxford Protein Production Facility and the X-ray structure determined by Dr Robin Owen at the Diamond Synchrotron. These co-crystal structures demonstrate that we have identified reagents that both directly and indirectly inhibit ligand binding to the receptor.
Our reagents provide a novel approach to reducing animal usage in antibody production. We have generated ABPs against numerous targets that have been used in Western blotting, ELISA and immunofluorescence.