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

Bijan Zakeri, Jacob O. Fierer, Emrah Celik, Emily C. Chittock, Ulrich Schwarz-Linek, Vincent T. Moy, Mark Howarth

Research output: Contribution to journalArticle

322 Citations (Scopus)

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 (Spy-Catcher) 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 languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number12
DOIs
StatePublished - Mar 20 2012

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Bacterial Adhesins
Protein Engineering
Peptides
Proteins
Streptococcus pyogenes
Thermodynamics
Fibronectins
Amides
Spectrum Analysis
Carrier Proteins
Buffers
Temperature

Keywords

  • Bacterial attachment
  • Chemical biology
  • Microbiology
  • Protein engineering
  • Single molecule biophysics

ASJC Scopus subject areas

  • General

Cite this

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. / Zakeri, Bijan; Fierer, Jacob O.; Celik, Emrah; Chittock, Emily C.; Schwarz-Linek, Ulrich; Moy, Vincent T.; Howarth, Mark.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 12, 20.03.2012.

Research output: Contribution to journalArticle

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