SpyCatcher

The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair.

Building upon this, SpyTag was obtained from CnaB2 by extracting the C-terminal beta strand containing the reactive aspartic acid at D556 and leaving the rest of the beta strands containing the reactive lysine K470 and the catalytic glutamic acid at E516 to become SpyCatcher, after further engineering to remove some hydrophobic surface residues.

[10][11][12][13] This enables fast production of vaccines as the central self-assembling molecule can be stocked up beforehand, whilst the antigen can be easily produced under optimal conditions to achieve proper protein folding.

Cyclized beta-lactamase, phytase, firefly luciferase, and xylanase (to name a few) have shown retained enzyme activity even after being subjected to heat at 100 °C.

[16] Subsequent modifications have enabled photo-responsive hydrogel formation,[17] user-defined control over cell-material interactions,[18] combined hyaluronan-elastin-like polypeptides,[19] as well creating protein scaffolds for enzyme flow biocatalysis.

Unlike SpyTag and SnoopTag which have extended structures, the region of RrgA used to create DogTag forms a β-hairpin and so predisposed for successful insertion into protein loops.

[23] DogTag has been successful coupled to DogCatcher when inserted into soluble proteins (superfolder GFP, HaloTag, and Gre2p).

Mutation of the catalytic glutamic acid residue (E77) in SpyCatcher to alanine stops isopeptide bond formation but does not prevent the initial non-covalent SpyTag/SpyCatcher association.

[27] SpyDock can be expressed in E. coli as soluble protein, purified using Ni-NTA and anion-exchange resins and immobilized to iodoacetyl-activated agarose through the unpaired cysteine introduced by the S49C substitution.

[27] Affinity to SpyTag003 has not been reported, but requires harsher conditions to ensure full dissociation suggesting it binds tighter.