Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

TLDR

Protein–peptide interactions typically exhibit low thermodynamic and mechanical stability, whereas the fibronectin‑binding protein FbaB from *Streptococcus pyogenes* contains a domain that forms a spontaneous isopeptide bond between lysine and aspartate. The study aimed to engineer a peptide (SpyTag) that rapidly forms a covalent amide bond with its protein partner (SpyCatcher) by splitting and rationally modifying the FbaB domain. The authors split the FbaB domain and engineered its fragments to create SpyTag and SpyCatcher, which can be fused at either terminus or internally and react specifically on mammalian cell surfaces. The SpyTag–SpyCatcher system reacts in high yield upon simple mixing across a wide range of pH, temperature, and buffer conditions, resists reversal by boiling or competing peptides, withstands forces up to 1 nN before covalent bond rupture, and offers a robust, irreversible linkage that could serve as a targetable lock in cells and a stable module for protein engineering.

Abstract

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.