Design and Evolution of SnoopLigase for Unbreakable Biomolecular Connections

Vaccination is one of the most effective ways to assure the health of humans and other animals and to ensure the security of the food supply. There is an urgent need to improve the way that vaccines are generated, because we lack effective vaccines to many of the most challenging diseases. Also, standard approaches to generate vaccines are slow compared to the speed that outbreaks of new diseases can occur. New vaccines are often developed from Virus-like particles (VLPs), which resemble viruses on the outside, but cannot cause disease because they do not have the virus genome inside. VLPs can be engineered to display a protein from a human or veterinary pathogen on their surface, to create an effective and safe vaccine. Decorating VLPs with such proteins from pathogens is a central problem for vaccine development. VLP decoration requires expensive trial-and-error experiments and has proved unfeasible with many protein targets. We have recently engineered a "bacterial superglue", termed SnoopLigase, which can catalyse the unbreakable linkage of one protein to another protein. This attachment is irreversible and applicable to a wide range of protein targets. SnoopLigase has proved useful in making enzymes more tolerant to harsh conditions and is likely to have diverse applications, including in assembly of diagnostic devices. In this proposal we will use evolution and computer-based design to engineer the next generation of SnoopLigase technology, so that it is faster, active in living cells (not just in the test-tube), and more potent at VLP decoration. SnoopLigase should be a general resource for scientists worldwide and speed up the creation of effective vaccines against major disease challenges.

Simple efficient reactions to connect biological building-blocks open up many new possibilities. We have designed SnoopLigase, a protein which catalyses specific isopeptide bond formation between two peptide tags. We initially developed these components by splitting the Streptococcus pneumoniae adhesin RrgA into three parts. Each part was then engineered based on structure, sequence homology, and computational prediction of stability. SnoopLigase demonstrated high yield coupling under a wide range of buffers and temperatures. Each tag was functional at the N- or C-terminus, while one tag was functional in loops. In search of a generic route to improve the resilience of enzymes, cyclisation via SnoopLigase allowed different enzymes to retain solubility and activity following heat treatment up to 100 C. In this project we will illuminate the mechanism of SnoopLigase catalysis. We will also develop novel selections for bond formation in bacterial and eukaryotic systems, towards dramatically enhanced activity of SnoopLigase reaction in cellular environments. The SnoopLigase catalytic activity is not present in nature and our initial version has exceptional affinity in staying bound to its ligated product. Through evolution and computational design, we will engineer SnoopLigase towards efficient multi-turnover catalysis. We will apply SnoopLigase for the urgent challenge of improving potency of vaccines against human and veterinary diseases. Our SpyTag/SpyCatcher strategy has enabled rapid and efficient decoration of antigens onto virus-like particles (VLPs), as vaccine candidates for a range of diseases. However, this technology is limited by the size of SpyCatcher hindering vaccine production and generating an unhelpful antibody response. With our engineered SnoopLigase, we will take on these challenges; to establish efficient and uniform VLP decoration, with tailored antigen orientation to focus the immune response on producing neutralising antibodies.

Who will benefit from this research?
Apart from academics, beneficiaries will include the Biotechnology Industry.
Biotechnology companies in diverse areas (e.g. agricultural enzymes, biofuels, cosmetics, diagnostics, therapeutics) have discussed with us licensing SpyTag/SpyCatcher. A particular area of interest, with patent filings of various companies, is assembly of robust devices for next generation sequencing of DNA and RNA. SnoopLigase only entered the public domain 3 months ago, but we are already talking to 3 companies about its licensing. However, for other companies we have spoken to, the innovations developed here will be crucial to SnoopLigase's industrial applicability. Developing this novel ligation approach will greatly extend the ability to assemble and control protein function. Products by these companies, harnessing covalent peptide-peptide ligation, 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 SnoopLigase and Tag variants should allow stable, simple and defined-orientation protein immobilisation in biosensors, nanopores and protein microarrays. Bridging protein antigens robustly to nanoparticles should optimise and accelerate vaccine assembly for veterinary and human diseases. SnoopLigase helps enzyme resilience and there have already been many applications of SpyTag/SnoopTag for orienting enzymes towards more efficient biotransformations. Demonstrating SnoopLigase activity in cells and enhancing turnover should advance these biotransformations, reducing the biosynthetic cost with a short peptide for ligation compared to the long Catcher domain. The new SnoopLigase and Tag 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 2 on phage-selected enhanced tags and a second patent in yr 3 on the multi-turnover SnoopLigase. The likely time-scale for commercial licensing of IP arising is from the 3rd 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
-development of presentation skills, through presenting within the University and at conferences, and discussing with Biotechnology companies
-taking the course "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 SnoopLigase 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 Ligase technologies, as we did previously for monovalent streptavidin in Nature Protocols. We will also provide rapid e-mail feedback as we have done for the large number of labs using SpyCatcher, SnoopCatcher and traptavidin.