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. agging with peptides (e.g., HA, myc, FLAG, His 6 ) is one of the most common ways to detect, purify, or immobilize proteins (1-4). Peptides are very useful minimally disruptive probes (5) but they are also "slippery"-antibodies or other proteins typically bind peptides with low affinity and poor mechanical strength (6-9). We sought to form a rapid covalent bond to a peptide tag without the use of chemical modification, artificial amino acids, or cysteines (disulfide bond formation is reversible and restricted to particular cellular locations).It has recently been found that Streptococcus pyogenes, like many other Gram-positive bacteria, contains extracellular proteins stabilized by spontaneous intramolecular isopeptide bonds (10). Here we explored the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein FbaB, found in invasive strains of S. pyogenes (11,12) and essential for phagocytosis-like uptake of the bacteria by endothelial cells (13). CnaB2 contains a single isopeptide bond conferring exceptional stability: CnaB2 remains folded even at pH 2 or up to 100°C (12). By splitting CnaB2 into peptide and protein fragments, followed by rational modification of the parts, we developed a peptide tag of 13 amino acids that rapidly formed a covalent bond with its protein partner (138 amino acids, 15 kDa) and characterized the conditions for reaction, cellular specificity of bond formation, and resilience of the reacted product.

Many natural biological systems - such as biofilms, shells and skeletal tissues - are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.

Peptides and synthetic peptide-like molecules are powerful tools for analysis and control of biological function. [1][2][3] One major problem with the use of peptides is the instability of their interactions with biomolecules, with typically micromolar affinity relating to the limited accessible surface area 4,5 and the intrinsic flexibility of peptides. 6 However, appending a short peptide tag is the most common way to allow a protein of interest to be isolated or detected, giving minimum perturbation to protein function. 7,8 Here we have designed a way to bind a peptide tag irreversibly, by adapting a recently discovered feature of amino acid chemistry: the spontaneous formation of an amide bond between a Lys and an Asn side chain in the appropriate environment. [9][10][11] Amide linkages outside of the protein main chain are termed isopeptide bonds (Figure 1A). Isopeptide bonds are chemically stable and resistant to most proteases. 12,13 Enzymes such as transglutaminases catalyze isopeptide formation, stabilizing the extracellular matrix and strengthening blood clots, 12 but these enzymes are large and have low sequence specificity. 12 Recently, certain proteins were discovered to autocatalyze single-turnover isopeptide bond formation, yielding ultrathin viral capsid chain mail, 9 or the proteolytically stable pili of Gram-positive bacteria, 10,11 through nucleophilic attack of the ε-amino group from a Lys to the Cγ group of an Asn, promoted by a nearby Glu (Figure 1B). 9,10 To apply spontaneous isopeptide bond formation to direct new covalent peptide interactions, we dissected the major pilin protein Spy0128 from Streptococcus pyogenes 10 (Figure 1C) and explored whether the two fragments would covalently associate. Split proteins have successfully reconstituted in many cases, including enzymes and fluorescent proteins, 14 albeit through noncovalent interactions. Spy0128 was split at the final β-strand of the C-terminal domain, to give the fragment pilin-C (Spy0128 residues 18-299, with N-terminal His 6 ) and the isopeptag (Spy0128 residues 293-308: TDKDMTITFTNKKDAE). This placed the reactive Asn on the isopeptag and the reactive Lys on pilin-C. To enhance recombinant expression in E. coli, the isopeptag was genetically fused to the N-terminus of maltose binding protein (MBP). To test covalent reaction, we mixed isopeptag-MBP and pilin-C, each at 10 µM, and boiled the samples in SDS before SDS-PAGE (Figure 1D). A new product formed at ∼80 kDa, consistent with reaction between isopeptag-MBP and pilin-C. We verified amide bond formation between isopeptag-MBP and pilin-C by mass spectrometry, demonstrating the loss of NH 3 upon reaction (Figure 2A). Pilin-C K179A, lacking the reactive Lys, did not form a covalent complex with isopeptag-MBP, determined by SDS-PAGE (Figure 1D) and mass spectrometry (Figure S1). Also, pilin-C did not react with MBP fused to an alternative peptide containing four potentially reactive Asn residues (MBP-isopeptag-N, Figure 1D).Spy0128 contains another isopeptide bond in its N-terminal...

Biotechnology is often limited by weak interactions. We suggest that an ideal interaction between proteins would be covalent, specific, require addition of only a peptide tag to the protein of interest, and form under a wide range of conditions. Here we summarize peptide tags that are able to form spontaneous amide bonds, based on harnessing reactions of adhesion proteins from the bacterium Streptococcus pyogenes. These include the irreversible peptide-protein interaction of SpyTag with SpyCatcher, as well as irreversible peptide-peptide interactions via SpyLigase. We describe existing applications, including polymerization to enhance cancer cell capture, assembly of living biomaterial, access to diverse protein shapes, and improved enzyme resilience. We also indicate future opportunities for resisting biological force and extending the scope of protein nanotechnology.

Many natural biological systems -such as biofilms, shells and skeletal tissues -are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and nonliving components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize selfassembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Additional information Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to T.K.L. Competing financial interests HHS Public AccessAuthor manuscript Nat Mater. Author manuscript; available in PMC 2014 November 01. Published in final edited form as:Nat Mater. 2014 May ; 13(5): 515-523. doi:10.1038/nmat3912. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptNatural multicellular assemblies such as biofilms, shells, and skeletal tissues have distinctive characteristics that would be useful for materials production and patterning 1-9 . They can detect external signals and respond via remodelling, implement patterning across different length scales, and organize inorganic compounds to create organic-inorganic composites. In this work, such systems provide inspiration for the design of environmentally responsive systems that can integrate biotic and abiotic materials via hierarchical self-assembly. To achieve these capabilities, we engineered artificial gene circuits and self-assembling amyloid fibrils together with synthetic cellular consortia 10-16 and abiotic materials.Our model system is curli, an extracellular amyloid material produced by E. coli that forms fibrils based on the self-assembly of the secreted major curli subunit CsgA 17 . Secreted CsgA monomers are templated on CsgB, which is anchored to the cell surface, to form curli fibrils; moreover, CsgA secreted from one cell can interact with CsgB on other cells 17 . Using synthetic riboregulators 18 , we implemented inducible transcriptional and translational control over the expressi...