Department of Biochemistry University of Oxford Department of Biochemistry
University of Oxford
South Parks Road
Oxford OX1 3QU

Tel: +44 (0)1865 613200
Fax: +44 (0)1865 613201
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Mark Howarth
Bionanotechnology and its Application to Cancer

Co-workers: Dr Michael Fairhead, Jayati Jain, Jacob Fierer, Gianluca Veggiani,
Christopher Schoene

Bionanotechnology involves manipulating and modifying components of living organisms, to generate tools on the 1-100 nanometre scale with desirable activities. Inspired by extraordinary molecular features from the natural world, our goal is to develop new approaches for investigating cellular function and for disease diagnosis.

Circulating tumour cells and engineering antibody technologies
Capturing the circulating tumour cells (CTCs) from blood samples is one of the most promising approaches to enable early diagnosis of cancer. CTC capture is already allowing rapid feedback on how a patient is responding to therapy and how the tumour is evolving. Existing magnetic CTC isolation approaches only capture the cells expressing high levels of tumour marker (EpCAM and HER2). We have shown how improving antibody affinity and cholesterol loading make it possible to recover low-expressing cancer cells. To improve further the isolation of CTCs, we are developing antibody technologies with new protein architectures and modes of target binding. This includes antibodies that form covalent bonds to endogenous protein targets.

Fluorescent nanoparticles to analyse receptors at the single molecule level
Being able to study molecules one at a time can give dramatic insights into the cell's function. We have developed new ways to target quantum dots (QDots) and conjugated polymer dots (Pdots), ultra-bright nanoparticles, to study cell surface receptors, in particular the type 1 insulin-like growth factor receptor (IGF1R) which is important in cell survival, differentiation and aging.

Nanohubs and the limits of protein-small molecule interaction strength
The interaction of streptavidin with biotin is one of the strongest non-covalent interactions in nature. Streptavidin is a central tool for bridging and purification in biological research, as well as showing success in clinical trials. We were able to make a version of streptavidin with 10-fold slower biotin dissociation. We have been investigating this interaction to understand mechanical strength of protein-ligand interactions, via single-molecule force spectroscopy and collisions with bacterial motors on DNA. We are also developing the use of streptavidin as an ultra-stable hub for nanoassembly.

Designing protein superglue from bacteria
Peptide tags are central to protein analysis and isolation, but they are also “slippery”- interactions to peptides are weak and reversible. We have harnessed and adapted the spontaneous formation of isopeptide bonds occurring in certain proteins of Streptococcus pyogenes. This enabled us to achieve an irreversible covalent bond between genetically-encoded protein (SpyCatcher) and peptide (SpyTag) partners; this bond is stable over time, at high temperatures, and against the forces in biological systems (blood flow, cell migration, molecular motors). We are extending the scope of this reaction to investigate force generation inside the cell and to create protein architectures for CTC isolation.

Publications

  1. Jain J, Veggiani G, Howarth M. (2013) Cholesterol loading and ultrastable protein interactions determine the level of tumor marker required for optimal isolation of cancer cells. Cancer Research 73(7):2310-21
  2. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109(12):E690-7
  3. Aleksic T, Chitnis M, Perestenko O, Gao S, Thomas P, Turner G, Protheroe A, Howarth M, Macaulay V. (2010) The type I IGF receptor translocates to the nucleus of human tumor cells. Cancer Research 70(16):6412-9
  4. Chivers CE, Crozat E, Chu C, Moy VT, Sherratt DJ, Howarth M. (2010) A streptavidin variant with slower biotin dissociation and increased mechanostability. Nature Methods 7(5):391-93
  5. Howarth M, Liu W, Puthenveetil S, Zheng Y, Marshall LF, Schmidt MM, Wittrup KD, Bawendi M, Ting AY. (2008) Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nature Methods 5(5):397-99
More Publications...

Research Images

Figure 1: Magnetic isolation of cancer cells

 



Figure 2: Enhancing one of the strongest non-covalent interactions in nature- crystal structure of traptavidin




Figure 3: Surviving a molecular car-crash. An ultra-stable variant of streptavidin (red and blue) survives collision by the motor protein FtsK (yellow and black) moving along biotinylated DNA at 5 kilobases per second


Figure 4: Cartoon of the conversion of an adhesion protein from pathogenic Streptococci into a genetically-encoded peptide tag which binds irreversibly


Contact: mark.howarth@bioch.ox.ac.uk
Graduate Student and Postdoctoral Positions: Enquiries with CV welcome
Website: http://users.ox.ac.uk/~bioc0756/MyWebs/activesite