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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.

Original publication

DOI

10.1073/pnas.1115485109

Type

Journal article

Journal

Proc Natl Acad Sci U S A

Publication Date

20/03/2012

Volume

109

Pages

E690 - E697

Keywords

Adhesins, Bacterial, Amides, Biophysics, Cell Membrane, Fibronectins, HeLa Cells, Humans, Hydrogen-Ion Concentration, Microscopy, Atomic Force, Molecular Sequence Data, Peptides, Protein Binding, Protein Engineering, Protein Structure, Tertiary, Spectrometry, Mass, Electrospray Ionization, Streptococcus pyogenes, Temperature