Plug-and-Play Streptavidin Hubs for Megaprotein Assembly and Nanoscale Control of Cell-Surface Triggering

There is strength in numbers, even at the molecular level. Biological molecules in clusters often have effects that cannot be achieved by those molecules in isolation. It is well known how to cluster one biological molecule into teams of 4, using a protein called streptavidin. Streptavidin is one of the most widely used tools in biological research and biotechnology because of this efficient and stable clustering. In this proposal we will establish how to engineer streptavidin to build a range of larger clusters containing precise numbers of molecules, up to 33. As well as determining the rules for how to build unbreakable clusters of different size and orientation, our new clusters will be powerful tools for studying and controlling many biological events. Signals are often communicated to cells through clustering of proteins at their surface. Killer T cells patrol the body, scrutinising the cells they pass to determine whether that cell is infected or healthy. According to the organisation of the clustering of receptors on the T cell, the T cell may kill the target cell or leave it unharmed. We will apply our different clusters to understand how the T cell makes this crucial decision.

Streptavidin is one of the most widely used tools in biological research and biotechnology, because of its rapid and stable capture of biotin. We have overcome two key challenges in the use of streptavidin: controlling the valency of biotin binding and providing an independent ultrastable interaction to streptavidin. SpyTag is a peptide we engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. We successfully fused SpyTag or SpyCatcher to subunits of the streptavidin tetramer, making "SpyAvidin" tetramers which bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. SpyAvidins provide a simple and robust hub so that distinct biomolecules can work together. SpyAvidins were also able to react together to form assemblies with higher valencies: octamers (8 subunits) or eicosamers (20 subunits). In this proposal we will address further challenges for plug-and-play biomolecular assembly. We will overcome the loss of biotin binding strength when streptavidin is chemically modified, thereby enhancing the stability and sensitivity of detection in flow cytometry and microscopy. Through fusing streptavidin with a protein covalently reacting with DNA, we will create a robust bridge between DNA and biotinylated ligands, as a simple and powerful way to enhance the functionality of DNA nanotechnologies. In addition, we will create a new form of protein architecture, streptavidin-based brush polymers consisting of up to 33 protein subunits. The precise and highly multivalent clusters generated in this research will be applied to dissect how nanoscale ligand organisation controls T cell receptor signalling. This proposal will create a range of simple-to-use tools, which should be valuable for diverse areas of biology, provide insight into clustering-dependent signalling, and establish principles for molecular construction at a new size and sophistication.

Who will benefit from this research?
Apart from academic scientists, the beneficiaries will include the Biotechnology Industry
Biotechnology companies in diverse areas have discussed with us about licensing or are currently evaluating Traptavidin and SpyTag/SpyCatcher. Developing these novel nanostructures will greatly extend the ability to assemble and control protein function. Products by these companies, harnessing covalent protein and peptide assemblies, should enhance detection speed and sensitivity for both individual proteins and cells in blood. Enhanced detection should have impact on disease 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 streptavidin variants should allow stable, simple and defined-orientation protein immobilisation in biosensors, columns for purification, and protein microarrays. Bridging protein reagents robustly to DNA should enhance immunoassays, through allowing multiplexed testing and enormous amplification of signals to improve sensitivity. The new streptavidin variants may directly comprise part of a commercial product/kit, or facilitate research leading to the generation of other products. Understanding the nanoscale regulation of T cell signalling will help to understand the effects of the T cell receptor in autoimmune disease, cancer and infectious disease and so may contribute to manipulation of T cell signalling in human and veterinary medicine, which is already a highly promising therapeutic target.

The likely time-scale for commercial licensing of IP arising is in the last 6 months of the award and the year 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 and applies them in cellular signalling
-development of presentation skills, through presenting within the University and at conferences, and discussing science and commercialisation with Biotechnology companies.
-taking the course in Entrepreneurship and Innovation at 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 Isis Innovation, who look after Oxford University IP, to ensure protection of all new IP arising. As we achieve key results, we will communicate with the University of Oxford press office and the BBSRC and with our existing industrial contacts, to ensure that significant findings are communicated to the public and potential industrial partners. We will publish detailed protocols in open access journals to facilitate adoption of the new multiprotein assembly technologies by other users, as we have done previously for the acceptor peptide and monovalent streptavidin in Nature Protocols. We will also provide rapid e-mail feedback as we have done for the large number of laboratories using BirA, traptavidin and monovalent streptavidin for diverse applications.