期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 12, 页码 E690-E697出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1115485109
关键词
bacterial attachment; chemical biology; microbiology; protein engineering; single molecule biophysics
资金
- Clarendon Fund
- National Institutes of Health
- National Science Foundation
- James and Esther King Biomedical Research Program
- School of Biology of the University of St. Andrews
- Oxford University Department of Biochemistry
- St. Peter's College
- New College
- Worcester College
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.
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