... understanding life in molecular detail

Dr Darren Tomlinson

Artificial binding proteins, protein protein interactions, oncology, diagnostics


Our interest lies in the development of novel scaffold proteins for presenting variable regions for molecular recognition (see lab webpage - http://dctomlinson.wixsite.com/laboratory). We have generated a highly complex library of binding proteins (termed Adhirons) that we screen using phage display to identify reagents for many different applications, including antibody replacements for immunoassays and diagnostics, and as reagents to study protein function by altering protein activity or inhibiting protein-protein interactions.

Current major projects include:
  • Inhibiting SH2 domains
  • Modulating MAPK signalling and Kras activity
  • Developing binding reagents to ion channels
  • Isolating diagnostics reagents
  • Inhibiting enzyme activity

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.

Figure 1. A. The ABP is shown in white, the loop regions are shown in grey. B. Differential scanning calorimetry to measure melting temperature of scaffold protein. C. Circular dichroism examining structures of the ABP and three random clones. D. Amino acid composition of the loop regions showing an even distribution with little bias.

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).

Figure 2. ABP targeting hSUMO2. A. An ABP raised against hSUMO2 was tested to determine specificity for hSUMO1 and hSUMO2 by ELISA. The hSUMO2 ABP specifically bound to hSUMO2, which was reflected in the binding affinity. B Blot showing the ability of the hSUMO2 ABP to inhibit RNF4’s SUMO-targeted ubiquitin ligase activity.

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

Fig3 Turbidity measurements to identify Adhirons that modify fibrin clot formation and lysis.

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.

Antibody replacements

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.

Figure 4. Co-crystals of two ABPs (white – red regions are the vairable regions) with a receptor extracellular domain (blue). The top co-crystal is showing indirect inhibition to ligand binding and the bottom crystal directly inhibits ligand binding.

 

Detailed research programme                  Close ▲
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University Academic Fellow



Postdoc (Leeds), 2002-2010

Astbury 7.108
School of Molecular and Cellular Biology
0113 343 7099
d.c.tomlinson@leeds.ac.uk
http://dctomlinson.wixsite.com/laboratory

Selected Publications

  1. Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications. Protein Eng Des Sel. 2014 May;27(5):145-55. doi: 10.1093/protein/gzu007. Epub 2014 Mar 25.

  2. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.  Nature Cell Biology 2015 Jul 13. doi: 10.1038/ncb3201. [Epub ahead of print]

  3. Phage Display Selected Magnetite Interacting Adhirons For Shape Controlled Nanoparticle Synthesis.. Chemical Science 2015, Advance Article

  4. Exploration of the HIF-1α/p300 binding interface using peptide and Adhiron phage display technologies to locate binding hot-spots for inhibitor development.  Molecular Biosystems, 2015 Jul 2. [Epub ahead of print]