SpySwitches: switchable SpyCatcher interactions yielding a modular toolbox for biochemistry and cell biology

Most of the processes that keep us alive are carried out by proteins. The majority of proteins do not act on their own but work in partnership with other proteins. Therefore, understanding how and where proteins interact is an important challenge. Some of these interactions only last for a fraction of a second, while others can last for days. We previously generated a "bacterial superglue" called SpyCatcher, which forms one of the strongest protein interactions ever found. SpyCatcher has found many uses, such as for accelerating creation of new vaccines or for building diagnostics to sequence DNA. In this proposal we will use evolution and design to engineer the next generation of SpyCatcher technology. We will generate new versions of SpyCatcher that span the full range of protein interactions, from one that breaks in seconds, another that breaks in minutes, all the way to the original that never breaks. We will also make versions of SpyCatcher that respond to acidic conditions or to light, so that researchers can turn the interaction on as desired. This unprecedented control over protein interactions should have many applications for basic research and biotechnology. In particular we will study how interactions in a cell change according to the mechanical stresses, as occurs when cells explore their surroundings or when a muscle contracts.

Simple efficient reactions for connecting biological building-blocks open up many new possibilities. We have designed SpyTag, a peptide which forms an unbreakable isopeptide bond upon encountering its protein partner SpyCatcher. SpyTag has enabled diverse applications, including for rapid assembly of potent vaccine candidates, multiplex functionalisation of antibodies, and generating ultra-stable enzymes. More recently we have generated Spy&Go, where an immobilised mutant SpyCatcher allows high purity capture of SpyTag fusions from bacterial and eukaryotic cells. Since SpyTag can enable purification, sensitive detection, irreversible anchoring, or programmable oligomerisation, we aim to make SpyTag a general resource to enhance the utility of proteins. In this project, we will establish a new direction for SpyTag, enabling its interaction to span the complete range of affinities and respond to external signals. Synthetic biology has huge potential but depends on the creation of modular units with reliable performance. Such programming has shown great advances at the level of RNA production and translation but there are limited resources to tune protein-protein interactions over orders of magnitude. This toolbox will be a unique resource for synthetic biology for control of how stably two proteins in the cell interact, along with providing precise spatial and temporal regulation. We will perform intensive biophysical investigation of the properties of the modified SpyCatcher toolbox and then establish switchable SpyCatcher as a resource to understand force propagation in live cells.

Who will benefit from this research?
Apart from academics, beneficiaries will include the Biotechnology Industry.
SpyTag/SpyCatcher has already been licensed by biotechnology companies in diverse areas (sequencing, diagnostics, vaccine assembly, antibody screening, agricultural enzymes, and cancer therapy). Modularity and tunable interactions are especially important to companies, who have pipelines for expressing thousands of protein targets for drug screening or tens of thousands of specific antibodies for interrogating biological responses. Therefore, we anticipate that the controlled affinity SpyCatcher003 interactions and efficient purification from the pH-switchable SpyCatcher003 variants would be beneficial in a range of industrial processes. Products by these companies, harnessing SpyTag modularity, should enhance detection speed and sensitivity in blood, while enhancing device resilience. Enhanced detection and more efficient/robust nucleic acid sequencing should have impact on diagnosis in animals and humans, beneficial to the general public, the farming community and the National Health Service.

How will they benefit from this research?
The new SpyCatcher003 variants should allow stable, simple and defined-orientation protein immobilisation in biosensors, nanopores and protein microarrays but now with the possibility of regenerating and reusing these devices. The new SpyCatcher003 variants may directly comprise part of a commercial kit, or facilitate research leading to the generation of other products.

We anticipate filing a patent in yr 1 on the tuned affinity series of SpyCatcher003 variants and a second patent in yr 3 on the pH-switchable SpyCatcher003. The likely time-scale for commercial licensing of IP arising is from the 2nd year of the award and the 3 years following the end of the award.

This project will provide important training for the postdoctoral researcher in:
-developing and executing a project which creates new tools by library-based evolution and computational design (Rosetta-based prediction of the effect of mutations on stability and pH-dependence)
-development of presentation skills, through presenting within the University and at conferences, and discussing with Biotechnology companies
-taking the course in Ideas to Impact at the Oxford University Business School
-assisting with protection of IP
-communicating their findings to a non-expert audience, including at the Oxfordshire Science Festival.

What will be done to ensure that they benefit from this research?
Publishing in high impact international journals is an effective way for us to communicate our findings to potential industrial partners. We will work with Oxford University Innovation (OUI), who look after Oxford University IP, to ensure protection of all new IP arising. OUI will contact potential commercial partners based on the network of companies already showing interest in our isopeptide technology (at least 30 published patents), as well as other companies relevant to the new applications that this SpyCatcher003 engineering makes possible. As we achieve key results, we will communicate with the University of Oxford press office and BBSRC, so significant findings are communicated to the public and potential partners. We will publish detailed protocols to facilitate adoption of the new SpyCatcher003 technologies, as we did for monovalent streptavidin in Nature Protocols and SpyTag in Meth Enz. We will continue to update at least monthly a "SpyInfo" webpage with links to all papers using SpyTag. On the same site, we will continue to update at least quarterly a "SpyBank" database with SpyTag and SpyCatcher sequences in papers or patents. This makes it easy to find related fusions and expression systems for new application. We will also provide rapid e-mail feedback to academic or industrial users, as we have done for the large number of labs using SpyTag, SnoopTag and monovalent streptavidin/traptavidin.