Peptide fusion tags are fundamental for the identification, detection, and capture of proteins in biological assays. Commonly used peptide fusion tags rely on temporary non-covalent interactions for binding, which can put constraints on assay sensitivity. Here, peptide fusion tags were developed that could specifically interact with protein binding partners via spontaneous and irreversible isopeptide bond formation. To develop covalently interacting peptide-protein pairs, outer-membrane proteins from Gram-positive bacteria that form autocatalyzed intramolecular isopeptide bonds were dissected to generate a short peptide fragment and a protein binding partner. Initially, the major pilin subunit Spy0128 from Streptococcus pyogenes was split to develop the 16 residue isopeptag peptide and the 31 kDa pilin-C protein partner. The isopeptag:pilin-C pair were able to react via spontaneous isopeptide bond formation between an Asn residue in isopeptag and a Lys residue in pilin-C without the requirement for any accessory factors, and with a yield of 60% after a 72 hr reaction. Reconstitution between the isopeptag:pilin-C pair was robust and occurred under all biologically relevant conditions tested, and also in the complex environment of a bacterial cytosol and on the surface of mammalian cells. A similar approach was also used to dissect the small CnaB2 domain that is part of the large FbaB fibronectin-binding protein from S. pyogenes. This led to the development of a more efficient peptide-protein pair, which was rationally modified to generate the highly optimized SpyTag:SpyCatcher pair. SpyTag is a 13 amino acid peptide with a reactive Asp that forms a spontaneous intermolecular isopeptide bond with a Lys present in the 12 kDa SpyCatcher binding partner. In a reaction with SpyTag, over 40% of SpyCatcher was depleted after 1 min and SpyCatcher could no longer be detected after 2 hr. The SpyTag and SpyCatcher reaction did not require any accessory factors and proceeded efficiently at a range of biologically relevant temperatures, pH values, concentrations, buffer compositions, and in the presence of commonly used detergents. The SpyTag:SpyCatcher technology was also used for specific cell surface labelling on mammalian cell membranes. SpyTag and SpyCatcher are both composed of the regular 20 amino acids and can therefore be genetically encoded as fusion constructs for a variety of in vitro and in vivo applications. Potential applications of the SpyTag:SpyCatcher technology include specific cell surface labelling, the development of novel protein architectures, and the covalent and irreversible capture of target proteins in biological assays.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:556121 |
| Date | January 2011 |
| Creators | Zakeri, Bijan |
| Contributors | Howarth, Mark |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:7d433538-0fa5-4081-acc5-06c1a77be322 |
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