Faculty of Biological Sciences

Dr Darren Tomlinson

PhD
UAF
School of Molecular and Cellular Biology

Contact:  Astbury 7.108, +44(0) 113 34 37099, email address for  

other websites -

astbury webpage

laboratory website

Research Interests

Protein engineering and utilising novel artificial binding 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 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. The scaffold protein is extremely stable with a Tm of 101oC and is the most stable ABP scaffold to date , and maintains the beta structure following loop insertion. 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. 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 Adhirons which differentiate between hSUMO1 and hSUMO2 isoforms. To confirm the ability to inhibit hSUMO2 binding they developed assays that test the Adhirons ability to inhibit SUMO interactions. In vitro recombinant RNF4 ubiquitinates polymers of hSUMO2 (poly-hSUMO22-8). Ads 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.

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. Some Adhirons 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 Adhirons against numerous targets that have been used in Western blotting, ELISA and immunofluorescence.


 

 

Faculty Research and Innovation



Studentship information

See also:

Modules taught

BIOC3160 - Laboratory/Literature/Computing Research Project
BIOL1303 - Introductory Skills for Biotechnology
BIOL2303 - Intermediate Skills for Biotechnology
BIOL3398 - Research Tools and Applications
BIOL3399 - Extended Research Project Preparation
BIOL5162M - Biopharmaceutical Drug Discovery
BIOL5269M - Commercialising Biopharmaceutical Products
BIOW5901X - Foundation module
BIOW5902X - Medicinal Chemistry and Drug Design

Dr Heather Martin  (Research Fellow)

Miss Anna Tang  (Research Technician)

Mr Thomas Taylor  (Research Technician)

Dr Christian Tiede  (Research Fellow)


Postgraduates

Danah Al-Qallaf (Primary supervisor) 40% FTE
Robert Bedford (Primary supervisor) 50% FTE
Lia De Faveri (Primary supervisor) 40% FTE
Sophie Heseltine (Primary supervisor) 70% FTE
Ajinkya Rao (Primary supervisor) 34% FTE
Anna Tang (Primary supervisor) 40% FTE
Katarzyna Zajac (Primary supervisor) 40% FTE
Modupe Ajayi (Co-supervisor) 30% FTE
Mansour Almutairi (Co-supervisor) 25% FTE
Irene Arrata (Co-supervisor) 40% FTE
Katherine Davies (Co-supervisor) 20% FTE
Katherine Kearney (Co-supervisor) 40% FTE
Zoe Lear (Co-supervisor) 33% FTE
Thanisorn Mahatnirunkul (Co-supervisor) 25% FTE
Joanna Mitchell (Co-supervisor) 30% FTE
Nnenna Nwogu (Co-supervisor) 30% FTE
Daniel Perez Witzke (Co-supervisor) 15% FTE
Rebecca Perrin (Co-supervisor) 20% FTE
Geoffrey Pettitt (Co-supervisor) 10% FTE
Sophie Roberts (Co-supervisor) 10% FTE
Fatma Salama (Co-supervisor) 50% FTE
Shazana Hilda Binti Shamsuddin (Co-supervisor) 25% FTE
Pattanapong Thangsunan (Co-supervisor) 25% FTE
Mark Waterhouse (Co-supervisor) 10% FTE