Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin
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 obtai...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 12; pp. E690 - E697 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences
20.03.2012
National Acad Sciences |
Series | PNAS Plus |
Subjects | |
Online Access | Get full text |
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Summary: | 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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Bibliography: | http://dx.doi.org/10.1073/pnas.1115485109 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 Author contributions: B.Z., J.O.F., E.C., U.S.-L., V.T.M., and M.H. designed research; B.Z., J.O.F., E.C., E.C.C., U.S.-L., and M.H. performed research; B.Z., J.O.F., E.C., U.S.-L., V.T.M., and M.H. analyzed data; and M.H. wrote the paper. 1B.Z. and J.O.F. contributed equally to this work. Edited by James A. Wells, University of California, San Francisco, CA, and approved January 17, 2012 (received for review September 21, 2011) |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.1115485109 |