Much of life’s complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide–protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen–deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide–protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.

SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram‐positive bacterial adhesin. In this work, we established a phage‐display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.

Peptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy&Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution. Spy&Go enabled sensitive purification of SpyTag-fusions from Escherichia coli , giving superior purity than His-tag/nickel-nitrilotriacetic acid. Spy&Go allowed purification of mammalian-expressed, N-terminal, C-terminal or internal SpyTag. As an oligomerization toolbox, we established a panel of SpyCatcher-linked coiled coils, so SpyTag-fusions can be dimerized, trimerized, tetramerized, pentamerized, hexamerized or heptamerized. Assembling oligomers for Death Receptor 5 stimulation, we probed multivalency effects on cancer cell death. Spy&Go, combined with simple oligomerization, should have broad application for exploring multivalency in signaling.

SpyTag is apeptide that forms aspontaneous amide bond with its protein partner SpyCatcher.T his protein superglue is ab roadly useful tool for molecular assembly,l ocking together biological building blocks efficiently and irreversibly in diverse architectures.W ei nitially developed SpyTag and SpyCatcher by rational design, through splitting ad omain from aG ram-positive bacterial adhesin. In this work, we established ap hage-displayp latform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under awide range of conditions and at either protein terminus.S pyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.Thousands of non-covalent protein-protein interactions mediate cellular function. However,e ngineering covalent interactions between protein partners brings ar ange of new opportunities for basic research and synthetic biology.[1] We have developed the use of spontaneous amide bond formation by peptide tags as asimple,specific, and genetically-encoded route to lock protein units together.[2] This technology, particularly the SpyTag/SpyCatcher pair, has been used in diverse applications including biomaterials,n ext-generation sequencing,e nzyme stabilization, and vaccine development. [1a, 3] Ak ey limitation has been relatively slow reaction at cellular expression levels.W ee stablished an evolutionary approach to achieve as econd-generation, faster-reacting version of this protein superglue.W et hen applied the enhanced properties for efficient and specific cell-surface functionalization, to investigate the outer-membrane dynamics of intimin, ap rotein relevant to human colonization by pathogenic bacteria.Since the SpyTag/SpyCatcher system is an unconventional approach to peptide interaction, it is likely that there are features of the interaction that cannot be predicted by rational design. Selection from phage libraries has been established for decades and the difficult thing is usually to detect weak interactions, [4] rather than the challenge of

Proteins can be empowered via SpyTag for anchoring and nanoassembly, through covalent bonding to SpyCatcher partners. Here we generate a switchable version of SpyCatcher, allowing gentle purification of SpyTagged proteins. We introduce numerous histidines adjacent to SpyTag’s binding site, giving moderate pH-dependent release. After phage-based selection, our final SpySwitch allows purification of SpyTag- and SpyTag003-fusions from bacterial or mammalian culture by capture at neutral pH and release at pH 5, with purity far beyond His-tag methods. SpySwitch is also thermosensitive, capturing at 4 °C and releasing at 37 °C. With flexible choice of eluent, SpySwitch-purified proteins can directly assemble onto multimeric scaffolds. 60-mer multimerization enhances immunogenicity and we use SpySwitch to purify receptor-binding domains from SARS-CoV-2 and 11 other sarbecoviruses. For these receptor-binding domains we determine thermal resilience (for mosaic vaccine development) and cross-recognition by antibodies. Antibody EY6A reacts across all tested sarbecoviruses, towards potential application against new coronavirus pandemic threats.