Power to the protein: enhancing and combining activities using the Spy toolbox

Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purification of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphene and the air/water interface. SpyTag/SpyCatcher is mechanically stable, so is widely used for investigating protein folding and force sensitivity. A toolbox of scaffolds allows SpyTag-fusions to be assembled into defined multimers, from dimers to 180-mers, or unlimited 1D, 2D or 3D networks. Icosahedral multimers are being evaluated for vaccination against malaria, HIV and cancer. For enzymes, Spy technology has increased resilience, promoted substrate channelling, and assembled hydrogels for continuous flow biocatalysis. Combinatorial increase in functionality has been achieved through modular derivatisation of antibodies, light-emitting diodes or viral vectors. In living cells, SpyTag allowed imaging of protein trafficking, retargeting of CAR-T cell killing, investigation of heart contraction, and control of nucleosome position. The simple genetic encoding and rapid irreversible reaction provide diverse opportunities to enhance protein function. We describe limitations as well as future directions.