title: Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
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.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Bijan
authors.initials: B
authors.lastname: Zakeri
authors.forename: Jacob O
authors.initials: JO
authors.lastname: Fierer
authors.forename: Emrah
authors.initials: E
authors.lastname: Celik
authors.forename: Emily C
authors.initials: EC
authors.lastname: Chittock
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwarz-Linek
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Moy
authors.forename: Mark
authors.initials: M
authors.lastname: Howarth
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism; D000577:Amides / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013297:Streptococcus pyogenes / Q000378:metabolism*; D013696:Temperature
chemical_list: D018829:Adhesins, Bacterial; D000577:Amides; D005353:Fibronectins; D010455:Peptides