![]() (C) Reaction rate of different Spy generations at low concentration. ![]() E77, also in stick format, transfers protons to facilitate reaction (based on PDB 2X5P and 4MLI). Reactive residues are marked in red in stick format. (B) Structural basis of reaction of SpyTag (cyan) with Sp圜atcher (dark blue). (A) Spontaneous isopeptide bond formation by reaction of Lys31 of Sp圜atcher with Asp117 of SpyTag (numbering from PDB 2X5P). 1,3 This approach allows specific covalent coupling of proteins both in vitro and in cells from various species. The reaction is irreversible and proceeds to >99% conversion. 2 Neither moiety contains any cysteine and so it is simple to use in different cellular locations. 1 SpyTag and Sp圜atcher can be genetically fused to the N- or C-terminus of proteins, and in some cases within internal loops of proteins. Spontaneous amidation between SpyTag/Sp圜atcher occurs in a wide-range of temperatures (4–37 ☌), buffers and pH values. Upon mixing, SpyTag and Sp圜atcher associate and spontaneously carry out an amidation reaction promoted by the Sp圜atcher residue Glu77, to form an intermolecular isopeptide bond ( Fig. The second part, dubbed SpyTag, is a 13-residue peptide that contains the reactive Asp117 ( Fig. Sp圜atcher is a 113-residue protein and contains the reactive Lys31. 1 CnaB2 spontaneously forms an intramolecular isopeptide bond between Lys31 and Asp117 ( Fig. Fundamentals of SpyTag/Sp圜atcher technology 1.1 Spontaneous amidation: kinetic and thermodynamic features SpyTag/Sp圜atcher is a protein coupling approach created by splitting the CnaB2 domain from the fibronectin binding protein FbaB from Streptococcus pyogenes. We describe limitations as well as future directions. The simple genetic encoding and rapid irreversible reaction provide diverse opportunities to enhance protein function. In living cells, SpyTag allowed imaging of protein trafficking, retargeting of CAR-T cell killing, investigation of heart contraction, and control of nucleosome position. Combinatorial increase in functionality has been achieved through modular derivatisation of antibodies, light-emitting diodes or viral vectors. For enzymes, Spy technology has increased resilience, promoted substrate channelling, and assembled hydrogels for continuous flow biocatalysis. Icosahedral multimers are being evaluated for vaccination against malaria, HIV and cancer. 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. SpyTag/Sp圜atcher is mechanically stable, so is widely used for investigating protein folding and force sensitivity. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphene and the air/water interface. 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. ![]() SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner Sp圜atcher, where both parts are genetically-encoded. ![]() Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Proteins span an extraordinary range of shapes, sizes and functionalities. ![]()
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