The Marvel of Covalent Bonds: Unlocking New Frontiers with protein “superglue” technology

The protein-protein interactions are measured in the equilibrium dissociation constant and in this regard an interaction with no dissociation and with high specificity which is challenging in a reaction with a typical equilibrium dissociation constant ranging from nanomolars to milimolars. This is especially important for scientists as the precision and the strength of these interactions. A solution to this challenge is to utilize covalent bonds which were shown to be versatile and stable which is called SpyTag/SpyCatcher System from Mark Howarth and his team in the University of Oxford.

A bacterial adhesin domain within the fibronectin-binding protein FbaB of Streptococcus pyogenes was identified to form a very durable intramolecular isopeptide bond between Lys and Asp residues (?). The Spytag/SpyCatcher System’s mode of action is based on splitting and engineering this domain that contains spontaneous isopeptide bond, that can couple with its counterpart with an amide bond. This allows a precise and specific lock in cells and a stable component for new protein designs (1115485109). The system has been developed as an interesting toolkit by utilizing the natural covalent bond forming function and further optimizing it for laboratory use which can be incorporated in genetic constructs and be expressed in many organisms.

This tool operates through a mechanism of formation of covalent bond between Lys residue in SpyTag and Asp residue of SpyCatcher which occurs when the short peptide sequence of a SpyTag encounters its protein couple SpyCatcher. A SpyTag can be fused to a target protein both at the N terminus, C terminus as well as in between and would form the covalent bond with SpyCatcher which similarly can be fused i.e. fluorescence proteins.

One major application is the construction of SpyRings, where SpyCatcher and SpyTag are fused to the ends of a protein, resulting in cyclization that strengthens protein against denaturation. This system is also used for the post-hydrogelation decoration of protein hydrogels, allowing target proteins to be stably attached to hydrogel materials. It can enable bioconjugation of target proteins to nanoparticles, integration into outer membrane vesicles, and the creation of proteinaceous nanocompartments like phage capsid proteins and virus-like particles. The SpyCLIP method uses SpyTagged RNA-binding proteins covalently attached to beads via SpyCatcher for pull-down assays. The system also facilitates fluorescent protein labeling for microscopy and the construction of artificial multi-enzyme nanodevices, which enhance enzymatic reaction efficiency, stability, and reusability.

Mark Howarth and his team later founded SpyBiotech company from the University of Oxford in 2017. By 2020 they worked on the development of a virus-like-particle (VLP) vaccine targeting COVID-19, and in 2021 they secured funding for advancing their novel vaccine technology platform with Phase I clinical study of its internal lead candidate, targeting human cytomegalovirus (HCMV), which is a betaherpesvirus that causes permanent infection in humans, and finally by July 2024 they announced the completion of enrollment in Phase I trials with positive results. References:
Hatlem, D. et al. (2019) ‘Catching a spy: Using the spycatcher-spytag and related systems for labeling and localizing bacterial proteins’, International Journal of Molecular Sciences, 20(9), p. 2129. doi:10.3390/ijms20092129.
Keeble, A.H. et al. (2019) ‘Approaching infinite affinity through engineering of peptide–protein interaction’, Proceedings of the National Academy of Sciences, 116(52), pp. 26523–26533. doi:10.1073/pnas.1909653116.
Zakeri, B. et al. (2012) ‘Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin’, Proceedings of the National Academy of Sciences, 109(12). doi:10.1073/pnas.1115485109.
https://spybiotech.com/spybiotech-announces-the-completion-of-enrollment-in-phase-i-trial-of-spyvlp01-targeting-human-cytomegalovirus-hcmv/