SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.

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.

SnoopLigase-Mediated Peptide-Peptide Conjugation and Purification.

Covalently linking together different proteins can enhance functionality for a range of applications. We have developed the SnoopLigase peptide-peptide conjugation method to easily and specifically link proteins fused to the peptides SnoopTagJr or DogTag via an isopeptide bond. SnoopLigase conjugation has been applied for enhancing enzyme resilience and for antigen oligomerization to enhance vaccine efficacy. Following conjugation, SnoopLigase and unreacted substrates can be removed by solid-phase immobilization of SnoopLigase, yielding purified protein-protein conjugates. Here, we describe procedures for designing tag-fused proteins, SnoopLigase purification, and ligation of SnoopTagJr and DogTag. We further define steps for the purification of the ligated product and quantification of ligation success.

Approaching infinite affinity through engineering of peptide-protein interaction.

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.

Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox.

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.