Proteins rarely work alone—they assemble into teams to carry out biological tasks, and this project will engineer a set of molecular superglues that let researchers stick specific proteins together on command, quickly and irreversibly, without unwanted cross-talk. The problem is that existing protein-linking tools are slow, unreliable in living cells, and cannot be used in parallel without pairs interfering with one another. This limits scientists’ ability to build multi-component protein machines for tasks such as assembling vaccine scaffolds, engineering cellular factories, or constructing synthetic biological circuits. If successful, the research will deliver a validated “rainbow” of Tag/Catcher pairs—each reacting only with its designated partner—that work rapidly inside bacteria, plants, and animal cells. This toolbox would give biologists a reliable way to program multi-protein assemblies, accelerating work in biomaterials, catalysis, cell therapy, and structural biology. The technology is already used in human clinical trials and livestock vaccine development, so improved versions could directly enhance vaccine production for farmed animals, supporting sustainable agriculture. The project is primarily fundamental science—it explores how split proteins can be computationally designed for fast, selective docking—but the resulting toolkit will be a transformative resource for the broader bioscience community.
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Biological function typically depends on proteins working together in teams. Routes for programming protein assembly should be specific and reliable in the context of many different proteins and cellular environments. Our group has generated a peptide called SpyTag and a protein called SpyCatcher (named after the bacterium S. pyogenes from which these parts were engineered). SpyTag/SpyCatcher technology has proven to be a powerful tool for bringing two proteins together simply, specifically and irreversibly. SpyTag technology has enabled diverse applications including in biomaterials, catalysis, cell therapy, structural biology and vaccines. SpyTag/SpyCatcher has been employed in bacteria, plants, mice, worms, flies, a range of livestock animals, and even in human clinical trials. Linking building-blocks irreversibly brings resilience over time and enhances the ability to monitor interactions in cells. We and others have now generated a set of Tag/Catcher pairs, harnessing the same idea of ligation through spontaneous covalent bond formation. However, these tools have low reaction speed, low ability as fusion partners and it is unknown whether the pairs are mutually specific and applicable in diverse cellular contexts. Here we will engineer a panel of Tag/Catcher pairs where each Tag or Catcher only reacts with its designated partner, ignoring other Tag/Catchers and other components of cells. This Tag/Catcher panel will provide a central underlying resource for the engineering of biology. This work will also establish fundamental insights into the computational design of split proteins for rapid reaction and cellular compatibility. Objective 1: Benchmarking the performance of the existing Tag/Catcher rainbow First we will quantify and compare key criteria for the large set of existing Tag/Catcher pairs, including reaction efficiency and selectivity. This objective will establish which pairs are best suited to take forwards to engineer for efficient building of molecular teams. Objective 2: Computational enhancement of Tag/Catcher pairing. Using a series of computational tools, we will design Tag/Catcher pairs with enhanced stability, reduced non-specific binding, and improved application in diverse cellular contexts. Objective 3: Directed evolution of rapid orthogonal Catcher toolbox. We will harness library selection to accelerate the reactivity, cellular display efficiency and selectivity. This objective will optimise Tag/Catcher pairs for selective and rapid reaction in a cellular context. Objective 4: Deep validation of optimised orthogonal Tag/Catcher pairs. Finally we will perform a complete characterisation of our optimised Tag/Catcher pairs, to demonstrate their applicability for the building of molecular teams in diverse cellular contexts. This objective will provide a thorough benchmarking, so that the Tag/Catcher panel can enable a new level of cellular programming and molecular assembly for the bioscience community. Rapid and selective multi-component coupling by this unique toolbox will have wide impact for both fundamental research and biotechnology. This proposal fits the BBSRC strategic challenge of “bioscience for sustainable agriculture and food”, given the wide use of Tag/Catcher pairs for assembly of vaccine candidates against diseases of farmed animals. This proposal fits BBSRC priorities of “understanding the rules of life” in investigating principles of split protein docking and isopeptide bond formation, and “transformative technologies” in the generation of a rapid and broadly applicable toolbox for molecular assembly.
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