Principal Supervisor: Dr Gemma-Louise Davies (Chemistry)
Co-supervisor: Dr Matthew I. Gibson (Chemistry), Dr Daniel Mitchell (Med School)
PhD project title: Responsive Magnetic Nanoparticle Based Protein Purification
University of Registration: University of Warwick
Protein expression underpins nearly all bioscience and biotechnology in terms of either application or understanding. A key challenge in this remains the isolation of the intact, folded proteins, usually with chromatography.
In this project, we will explore the use of responsive magnetic nanoparticles as alternatives to a traditional stationary phase. Particular advantages of this approach include the huge range of potential functionality which can be embedded on the particles to enable both traditional binding motifs (such as oligo-histidine and biotin tags) but also new ones based on reversible covalent immobilisation such as sortase-mediated ligation. Finally, the purification of high-value carbohydrate binding proteins, particularly lectins, but also antibodies, will be achieved by this method, which is currently challenging, contributing to their high cost and slow translation as therapeutics or drug targets.
The use of particles also maximises accessible surface area for protein capture. By using magnetism as the isolation route, little or no infrastructure is required, and the particles will be fully reversible. A dynamic polymer coating on the nanoparticle surface will prevent non-specific interactions (which is the prime reason for impurities in current methods) and also enable the particles to be recycled for repeated usage. A unique feature of this will be the capture of metabolically labelled proteins and glycans, removing the need for any protein engineering.
By using established, rigorous synthetic procedures,1 this project will focus on the isolation of high value, challenging proteins, not on the synthesis of the particles, and is therefore very focused on the bioscience.
A schematic of the particles, with reversible capture motifs is shown below.
Projected timescales for this project are summarised below, showing the rapid translation to purification of ‘real’ and relevant new proteins.
Month 6 – Library of diversely labelled magnetic particles obtained.
Month 12 – Reversible capture, release and assessment of purity (HPLC/FPLC) of enriched proteins demonstrated from complex, relevant solutions.
Month 18 – Capture and isolation of multiple human lectins from recombinant expression systems.
Month 24 – Capture of metabolically labelled proteins, and subsequent glycosidase mediated release.
Month 36 – Scale up to demonstrate on a larger scale (i.e. not micrograms).
Project Management Team
GLD will lead the project, and has experience of functional nanoparticle use, including their application as magnetic separation devices, as well as fabrication of nanoparticles, ensuring that this project is focussed on the biotechnological processes, and not hindered by synthetic method development. GLD’s extensive experience working on cross-disciplinary projects will ensure objectives are delivered on time. Her research lies at the interface with chemistry, biology and medical sciences. MIG has extensive expertise in glycan recognition and will advise on this aspect of the project. GLD and MIG have a strong collaborative relationship and have published several papers together. Recent collaborations between MIG and DM (WMS) have resulted in high impact papers (e.g. Nature. Commun., 2014, 5, 3244).
S. McCarthy, G.-L. Davies and Y. K. Gun’ko, Nature Protocols, 2012, 7, 1677-1693.
BBSRC Strategic Research Priority: Bioenergy and industrial biotechnology
Techniques that will be undertaken during the project:
- Protein expression and sequencing
- SPR analysis of binding affinities to prove function
- Circular dichroism
- Magnetic nanoparticle synthesis and characterisation using biophysical methods including, dynamic and static light scattering, electron microscopy and UV-Vis spectroscopy
Contact: Dr Gemma-Louise Davies, University of Warwick