Biomimetic Crystallisation of Metal-Organic Materials for Protein Isolation and Stabilization

Proteins have evolved over hundreds of years to perform a wide variety of highly sophisticated tasks, however their structural fragility continues to limit their application beyond biological settings. Development of an encapsulation strategy capable of supporting the three-dimensional structure of proteins that often gives rise to their desirable properties, is therefore essential if the full potential to utilise proteins is to be realised. Specifically, benefits arising from protein stabilisation are foreseen in:
i) the manufacturing sector where enzymes may be able to replace expensive and environmentally toxic catalysts currently in use,
ii) in healthcare where purification and instability issues raise the cost of protein pharmaceuticals limiting their application for long-term disease management and their use in the Third World, and also
iii) in research science where biologists strive to understand how enzymes function within confined spaces.

This proposal will systematically evaluate the materials properties most suited for encapsulating and stabilising proteins in their native state. A biologically inspired crystallisation methodology is proposed where metal-organic frameworks (MOFs) assemble around proteins in solution, thus avoiding limitations associated with the synthesis and stability of MOFs with sufficiently large internal void pockets to accommodate proteins. A wide variety of conditions will be screened in the initial stages of the project to evaluate the metal, organic ligand, crystallisation conditions and protein properties best suited to biomimetic crystallisation of MOFs without negatively impacting the protein. Encapsulation of purified proteins as well as those introduced in complex biological mixtures will be evaluated.

Following optimisation of the crystallisation conditions a range of enzymes will be encapsulated and changes in the stability and rate of enzymatic reaction for the encapsulated enzyme versus the non-encapsulated enzyme will be evaluated. It is hypothesised that encapsulation will increase the stability of the enzyme, but may reduce or modify the rate of the catalysed reaction and the reaction scope due to limitations associated with the movement of molecules through the framework material.

Finally, detailed characterisation of the protein within the restricted space of the stabilising framework material will be undertaken and potential opportunities for characterisation of biological proteins that typically display a high degree of disorder or those that are known to operate within a confined space will be evaluated.

Technology that allows translation of highly evolved proteins to non-natural environments has the potential to revolutionise the vast majority of chemical processes, by enabling replacement of small, synthetic molecules with biomacromolecules that display exquisite sensitivity and selectivity under ambient conditions. One promising strategy for stabilisation of proteins outside of their natural environment is encapsulation within molecular containers capable of supporting the fragile three-dimensional structure that is often essential for their desirable properties.

Specifically we have identified the industrial and medicinal biotechnology sectors as areas that would directly benefit from protein encapsulation strategies. Encapsulation of industrially useful enzymes offers opportunities to increase their recyclability and thus cost effectiveness, as well as providing a strategy by which enzymes could be effectively used in organic solvents. Similarly, encapsulation provides stabilisation benefits for the emerging class of protein pharmaceuticals or biologics. Biologics have demonstrated greater activity and selectivity over traditional small molecule therapeutics and provide treatment options for some diseases where there are currently no traditional small molecule drug treatments available. Limitations associated with purification of bulk protein and stabilisation for medium to long-term storage do however continue to limit the development and wide-spread applicability of many biologics especially for the treatment of chronic illnesses or in the Third World, where maintaining strict storage conditions may not be possible.

Maximal economic impact will thus be achieved through engagement with relevant industrial stakeholders, to ensure that solutions we seek to develop are aligned with their needs, and to ensure the most relevant challenges are addressed in a timely manner. All industrially relevant IP will be managed through consultation with the University of Manchester Intellectual Property Office who have extensive experience of licensing technologies on a local, national and international platform.

In addition to the industrial benefactors, the academic impact of this research will be realised through dissemination of results in high-impact international journals and research presentations. The interdisciplinary nature of the research is likely to benefit wide sectors of the research community from those interested in fundamental biological properties to those working in materials science. Additional societal impact will be gained through media interest and press releases explaining the value of our published work pertaining to real-world applicability. These public engagement opportunities will be complemented by outreach activities chosen to appeal to different sectors of society from school children through to adults.

Finally the interdisciplinary nature of this project will undoubtedly support the training and development of the PDRA and PhD student working on the research. The experience gained by these researchers, alongside their tailored personal development will ensure they are exceptionally qualified for research jobs at the interface of materials chemistry and bioscience, thus presenting opportunities for them in both industrial and academic settings.