SpyTag Peptide with Alkoxyl Aspartic Acids for pH-Dependent Activation of the SpyCatcher/Tag System.

The SpyCatcher/SpyTag system is a protein pair that forms a covalent isopeptide bond without an additional energy supply. The ability to connect fused proteins makes this system an attractive tool for several protein engineering applications. Conditional activation of the SpyCatcher/SpyTag complex formation further expands the use of this system. Here, we evaluated the pH activation of SpyTag using alkoxyaspartic acids in the isopeptide-forming residue. We found that a peptide with an ethoxy group can be activated by hydrolysis under high pH conditions. However, the hydrolysis induces isoaspartate (isoAsp) formation, which is confirmed by an isoAsp-inserted short peptide. We overcame this problem by changing the C-terminal side of the aspartic acid position to Pro, which does not form isoAsp under high pH conditions. The findings of this study provide fundamental knowledge of the synthetic construction of the modified SpyTag peptide.