Faculty of Biological Sciences

Dr Darren Tomlinson

PhD
Bioscreening Technology Group Manager
School of Molecular and Cellular Biology

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

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.

other websites -

http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=DT

 

Faculty Research and Innovation


Studentship information

See also:

Modules taught

BIOC3160 - Lab/Lit/Comp Research Project
BIOL1303 - Introductory Skills for Biotechnology
BIOL5292M - Bioscience MSc Research Proj
BIOL5294M - MSc Bioscience Res Project Pro

Tiede C; Tang AA; Deacon SE; Mandal U; Nettleship JE; Owen RL; George SE; Harrison DJ; Owens RJ; Tomlinson DC; McPherson MJ Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications. Protein Eng Des Sel 27 145-155, 2014
DOI:10.1093/protein/gzu007
View abstract

King RJ; Tiede C; John V; Trehan A; Tomlinson D; Ajjan RA Inhibiting complement C3 and fibrinogen interaction: a potential novel therapeutic target to reduce cardiovascular disease in diabetes DIABETIC MEDICINE 31 57-58, 2014

Adams M; Cookson VJ; Higgins J; Martin HL; Tomlinson DC; Bond J; Morrison EE; Bell SM A high-throughput assay to identify modifiers of premature chromosome condensation. J Biomol Screen 19 176-183, 2014
DOI:10.1177/1087057113495443
View abstract

Fearnley GW; Smith GA; Odell AF; Latham AM; Wheatcroft SB; Harrison MA; Tomlinson DC; Ponnambalam S Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells. Methods Enzymol 535 265-292, 2014
DOI:10.1016/B978-0-12-397925-4.00016-X
View abstract

Stadler LK; Tomlinson DC; Lee T; Knowles MA; Ko Ferrigno P The use of a neutral peptide aptamer scaffold to anchor BH3 peptides constitutes a viable approach to studying their function. Cell Death Dis 5 e1037-, 2014
DOI:10.1038/cddis.2013.564
View abstract

Martin HL; Adams M; Higgins J; Bond J; Morrison EE; Bell SM; Warriner S; Nelson A; Tomlinson DC High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers. PLoS One 9 e88338-, 2014
DOI:10.1371/journal.pone.0088338
View abstract

Martin HL; Smith L; Tomlinson DC Multidrug-resistant breast cancer: Current perspectives Breast Cancer: Targets and Therapy 6 1-13, 2014
DOI:10.2147/BCTT.S37638
View abstract

Nash CE; Holliday DL; Mavria G; Tomlinson D; Hanby A; Berditchevski F; Speirs V Development and Characterisation of a 3D Tri-Culture Model of Normal Breast as a Tool for Cancer Initiation Studies Shows Overexpression of Her2 and Her3 Alters 3D Epithelial Architecture, 2013

King R; Tiede C; McPherson M; Manfield I; Fishwick C; Tomlinson D; Ajjan R A novel methodology for modulation of fibrin clot lysis: the role of fibrinogen-targeted artificial binding proteins JOURNAL OF THROMBOSIS AND HAEMOSTASIS 11 721-721, 2013

Tomlinson DC; Knowles MA; Speirs V Mechanisms of FGFR3 actions in endocrine resistant breast cancer International Journal of Cancer 130 2857-2866, 2012

Tomlinson DC; Baxter EW; Loadman PM; Hull MA; Knowles MA FGFR1-induced epithelial to mesenchymal transition through MAPK/PLCγ/COX-2-mediated mechanisms. PLoS One 7 e38972-, 2012
DOI:10.1371/journal.pone.0038972
View abstract

di Martino E; Tomlinson DC; Knowles MA A Decade of FGF Receptor Research in Bladder Cancer: Past, Present, and Future Challenges. Adv Urol 2012 429213-, 2012
DOI:10.1155/2012/429213
View abstract

Stadler LK; Hoffmann T; Tomlinson DC; Song Q; Lee T; Busby M; Nyathi Y; Gendra E; Tiede C; Flanagan K; Cockell SJ; Wipat A; Harwood C; Wagner SD; Knowles MA; Davis JJ; Keegan N; Ferrigno PK Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant. Protein Eng Des Sel 24 751-763, 2011
DOI:10.1093/protein/gzr019
View abstract

Lamont FR; Tomlinson DC; Cooper PA; Shnyder SD; Chester JD; Knowles MA Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo. Br J Cancer 104 75-82, 2011
DOI:10.1038/sj.bjc.6606016
View abstract

Tomlinson DC; Knowles MA Altered splicing of FGFR1 is associated with high tumor grade and stage and leads to increased sensitivity to FGF1 in bladder cancer. Am J Pathol 177 2379-2386, 2010
DOI:10.2353/ajpath.2010.100354
View abstract

Hafner C; Di Martino E; Pitt E; Stempfl T; Tomlinson D; Hartmann A; Landthaler M; Knowles M; Vogt T FGFR3 mutation affects cell growth, apoptosis and attachment in keratinocytes. Exp Cell Res 316 2008-2016, 2010
DOI:10.1016/j.yexcr.2010.04.021
View abstract

Tomlinson DC; Lamont FR; Shnyder SD; Knowles MA Fibroblast growth factor receptor 1 promotes proliferation and survival via activation of the mitogen-activated protein kinase pathway in bladder cancer. Cancer Res 69 4613-4620, 2009
DOI:10.1158/0008-5472.CAN-08-2816
View abstract

Tomlinson DC; Knowles M; Speirs V ACTIVATION OF FGFR3 INDUCES RESISTANCE TO ENDOCRINE THERAPY IN MCF-7 CELLS, 2009

Kotwal S; Tomlinson D; Paul AB; Knowles MA ERBB3 OVER EXPRESSION IN BLADDER CANCER CELLS LEADS TO ENHANCED MOTILITY AND INVASION EUR UROL SUPPL 8 198-198, 2009

Hurst CD; Tomlinson DC; Williams SV; Platt FM; Knowles MA Inactivation of the Rb pathway and overexpression of both isoforms of E2F3 are obligate events in bladder tumours with 6p22 amplification. Oncogene 27 2716-2727, 2008
DOI:10.1038/sj.onc.1210934
View abstract

Tomlinson DC; Baldo O; Harnden P; Knowles MA FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer. J Pathol 213 91-98, 2007
DOI:10.1002/path.2207
View abstract

Tomlinson DC; Hurst CD; Knowles MA Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer. Oncogene 26 5889-5899, 2007
DOI:10.1038/sj.onc.1210399
View abstract

Tomlinson DC; L'Hôte CG; Kennedy W; Pitt E; Knowles MA Alternative splicing of fibroblast growth factor receptor 3 produces a secreted isoform that inhibits fibroblast growth factor-induced proliferation and is repressed in urothelial carcinoma cell lines. Cancer Res 65 10441-10449, 2005
DOI:10.1158/0008-5472.CAN-05-1718
View abstract

Jebar AH; Hurst CD; Tomlinson DC; Johnston C; Taylor CF; Knowles MA FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene 24 5218-5225, 2005
DOI:10.1038/sj.onc.1208705
View abstract

Tomlinson DC; Grindley JC; Thomson AA Regulation of Fgf10 gene expression in the prostate: identification of transforming growth factor-beta1 and promoter elements. Endocrinology 145 1988-1995, 2004
DOI:10.1210/en.2003-0842
View abstract

Freestone SH; Marker P; Grace OC; Tomlinson DC; Cunha GR; Harnden P; Thomson AA Sonic hedgehog regulates prostatic growth and epithelial differentiation DEV BIOL 264 352-362, 2003
DOI:10.1016/j.ydbio.2003.08.018