SpyTagSpyCatcher

SpyTagSpyCatcher is a bioconjugation system that enables spontaneous, covalent linkage between two protein fragments. It is derived from a split domain of a bacterial adhesion protein and engineered to reconstitute when the SpyTag and SpyCatcher components meet. SpyTag is a short peptide (about 13 amino acids) and SpyCatcher is a compact protein fragment (about 133 amino acids). When fused to target proteins, peptides, or materials, SpyTag and SpyCatcher readily form a covalent bond under physiological conditions without enzymes, resulting in a stable linkage.

Mechanism and properties: The reaction forms a covalent isopeptide bond between a residue on SpyCatcher and a residue on SpyTag, proceeding rapidly and selectively with high specificity. The bond is irreversible under typical conditions, and the reaction is tolerant of a range of pH and temperatures, making it versatile for in vitro and in vivo applications.

Applications: The system is used for modular protein conjugation, labeling, and the assembly of multi-domain or multi-component protein complexes. It enables covalent linkage of proteins to surfaces, nanoparticles, and materials, facilitating the construction of biomaterials, diagnostic assays, imaging tools, and immobilized affinity systems. It supports both simple attachments and more complex, multi-step assemblies through sequential or orthogonal tag/catcher pairs.

Variants and considerations: Since its introduction, several variants have been developed to improve reaction kinetics, stability, or compatibility with different assays. Designers consider tag/catcher orientation, linker length, and tag accessibility to optimize ligation efficiency. Limitations include the irreversible nature of the bond and potential steric or functional disruption if fusion points are poorly positioned or immunogenic in therapeutic contexts.