Engineered SpyTag002 Peptide Improves Reaction Rate

Example 5

The SpyTag002-MBP fusion has a reaction rate of 0.40 μM⁻¹ min⁻¹ with SpyCatcher002. We surprisingly determined that the reaction rate could be further improved by introducing additional modifications to the SpyTag002 peptide.

Substitution of the threonine residue at position 3 of SpyTag002 (SEQ ID NO: 3) with histidine, i.e. reversion to the residue at the equivalent position in SpyTag, resulted in a peptide (SEQ ID NO: 4) with a reaction rate of 0.53-0.55 μM⁻¹ min⁻¹, i.e. about a 35% increase in activity (FIG. 15A).

Modification of the improved peptide to include arginine and glycine residues at the N-terminus (SEQ ID NO: 5) more than doubled the reaction rate to 1.21 μM⁻¹ min⁻¹ (FIG. 15B).