Protein team assembly for controlling cell signalling and catalysis

SnoopTag/Snoop Catcher is a genetically-encoded peptide/protein pair that reacts to form an irreversible isopeptide bond simply upon mixing. This reaction makes it possible to link proteins into chains on a solid phase in a highly specific and efficient manner. The method is simple to carry out, modular and produces irreversibly linked and molecularly-defined protein chains. Proteins often work in teams and so this method creates the opportunity to either control or artificially generate such multi-component systems. Applications include regulating immune responses and cell differentiation, or generation of antibody polymers for sensitive diagnostics, multi-enzyme assemblies to enhance metabolic flux, and strong modular biomaterials.

SnoopTag

Researchers at the University of Oxford recently developed a ‘bacterial superglue’, known as SpyTag/SpyCatcher, from Streptococcus pyogenes. They have now developed a new peptide/protein pair named SnoopTag/SnoopCatcher, from Streptococcus pneumoniae, which spontaneously locks together through a covalent bond. SnoopTag/SnoopCatcher reaction is high yielding and fast while the bond can survive extreme pH, high ionic strength and detergents. SnoopTag/SnoopCatcher and SpyTag/SpyCatcher are genetically encodable and are mutually unreactive, so they allow many new opportunities for controlled and irreversible linkage of peptide and protein components. By exploiting these peptide/protein pairs together, a modular and high yielding approach to irreversibly assemble proteins into chains has been developed.

Generating protein chains

Solid-phase synthesis, which involves reactants bound to resin, enabled a revolution in the generation of peptides and oligonucleotides to efficiently explore and control biological function. However, solid-phase linkage for proteins is much more complex because of a large number of potential reactive groups present. Previous methods only generated short protein chains because the methods give a range of undesired side-products, have weak linkages, or use non-genetically-encoded components.

The key properties of SnoopTag/SnoopCatcher-based solid-phase assembly are:

  • Simple add-and-wash procedure
  • Irreversible linkage
  • Completely genetically encoded, with no use of alternative amino acids
  • High specificity
  • No need to purify intermediates
  • Protein unit to be added modified only with two small peptide tags, which can be located at the N-terminus, C-terminus or an internal site on the protein
  • No cysteines in the reaction, so applicable to proteins containing free cysteines or disulfide bonds

This approach enables combinatorial assembly of polyprotein teams and should open up a new area for controlling how protein components work together. Uses of this invention include vaccine generation, enzyme substrate channelling, antibody polymerisation, drugs for activating cell signalling, and biomaterials.

This technology is subject to a patent application. Oxford University Innovation is interested in hearing from potential partners who wish to develop the technology and explore the commercial opportunities.

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