Laccase-catalyzed bioconjugation of tyrosine-tagged functional proteins.

The site-specific cross-linking of functional proteins creates macromolecular assemblies that exhibit unique biochemical and/or physicochemical properties. Herein, we explored the potential of laccase as a biocatalyst for the site-specific cross-linking of tyrosine-tagged proteins. Trametes sp. laccase (TL) was selected as the cross-linking catalyst, and Escherichia coli alkaline phosphatase (BAP) and antibody-binding proteins (pG2 pAs) were employed as model proteins. The protein models were genetically fused to a peptide tag containing a tyrosine residue (Y-tag) at the N- and/or C-termini. Proteins without Y-tags were used as controls. The Y-tagged proteins could be recognized by TL as macromolecular substrates, leading to the oxidative formation of protein polymers, whereas no polymerization was observed with intact BAP or pG2 pA. The TL-catalyzed cross-linking of Y-tagged proteins proceeded at a relatively high pH in comparison with that of small phenolic substrates. Co-polymers of BAP and pG2 pA were able to be prepared by mixing the aqueous solution of each component in the presence of TL. A combination of bis-Y-tagged pG2 pA (Y-pG2 pA-Y) and Y-tagged BAP (BAP-Y) yielded functional co-polymers compatible with enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA of ovalbumin with anti-OVA IgG depended on the molar ratio of BAP-Y and Y-pG2 pA-Y in the TL-catalyzed cross-linking reaction. A high molar ratio of BAP-Y to Y-pG2 pA-Y (75:1) resulted in the highest absorbance in the ELISA. The results suggested that the formation of a bifunctional protein polymer with a high molar ratio of signaling unit to antibody-binding unit gave better performance in antigen detection than using lower ratios.