- Position: Associate Professor
- Areas of expertise: Biomolecular engineering; cancer; cell signalling; cell biology; antimicrobial resistance
- Email: D.C.Tomlinson@leeds.ac.uk
- Phone: +44(0)113 343 7099
- Location: 7.108 Astbury
- Website: Laboratory website
I studied for a PhD in developmental biology at the Univeristy of Edinburgh in the MRC Reproductive Biology Unit. After completing my PhD I moved to Leeds and worked as a PDRA in Prof Margaret Knowles's lab studying the function of growth factor receptors in bladder cancer.
In 2010 I set up two facilities in Leeds - a siRNA screening facility and a facility to produce non-antibody binding proteins. In 2015 I became a University Academic Fellow at Leeds to build a research group to fully exploit the use of Affimers for studying protein function and in 2018 I was promoted to Associate Professor.
Protein engineering and utilising Affimer proteins in molecular biology
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 numerous alternatives are being developed including protein, RNA and DNA aptamers. These alternatives can bind to 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 Affimer library. The Affimer platform is based on two constant small 91 amino acid scaffold proteins that constrains one or two randomised nine amino acid loop regions for molecular recognition. The scaffold protein is extremely stable with a Tm of 101oC , and maintains the beta structure following loop insertion. We have developed a large naïve phage display library (>3x1010) of Affimer reagents 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. Our laboratory mainly uses Affimer reagents to develop novel biologics and to study protein function in a broad range of diseases including antimicrobial resistance and cancer.
We also work collaboratively with academics, clinicians and industrial partners on numerous projects. A few examples are listed below
Inhibiting protein-protein interactions
We have previously demonstrated that we can generate specific Affimer reagents to block human SUMO2 (hSUMO2), and for the first time we have developed reagents which differentiate between hSUMO1 and hSUMO2 isoforms. To confirm the ability to inhibit hSUMO2 binding we developed assays that test the Affimers ability to inhibit SUMO interactions. In vitro recombinant RNF4 ubiquitinates polymers of hSUMO2 (poly-hSUMO22-8). Affimers specific for GFP (irrelevant control) and for hSUMO1 were unable to inhibit RNF4s ability to ubiquitinate poly-hSUMO22-8, whereas Affimer reagents specific for hSUMO2 robustly inhibited this activity at less than 1 µM.
We are now using Affimer reagents to study protein-protein interactions in the MAPK pathway. This pathway is dysregulated in many diseases including cancer, yet reagents that can specifically inhibit protein interactions are lacking adn therefore the basic understanding of what each of these domains is lacking. We have isolated Affimer reagents against numerous domains of proteins in this pathway, including Grb2, Sos and Ras. These Affimers are now being characterised in cell based assays to assess changes in cellular phenotypes
Inhibiting protein function
We are working with Dr Ramzi Aijan to modulate blood clot formation or facilitate lysis (clot breakdown). We raised numerous Affimer reagents against fibrinogen and used assays to assess changes in fibrin clot formation and lysis. Many adhirons either prolonged clot formation or altered lysis. Some Affimers 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 Affimers that block receptor function (TNF release and phagocytosis). Three of the Affimer 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 Affimers against numerous targets that have been used in Western blotting, ELISA and immunofluorescence. One of our papers was recently highly commended by the NC3Rs for reducing animal usage in research.<h4>Research projects</h4> <p>Any research projects I'm currently working on will be listed below. Our list of all <a href="https://biologicalsciences.leeds.ac.uk/dir/research-projects">research projects</a> allows you to view and search the full list of projects in the faculty.</p>
- Imaging the interactome
- Multiscale analysis of extracellular regulation of BMB signalling
- Understanding how proteins are organised in the Z-disk, a super-resolution approach
- Faculty Graduate School
- FindaPhD Project details: