Laccase-catalyzed bioconjugation of tyrosine-tagged functional proteins

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Abstract

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 (pG2pAs) 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 pG2pA. 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 pG2pA were able to be prepared by mixing the aqueous solution of each component in the presence of TL. A combination of bis-Y-tagged pG2pA (Y-pG2pA-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-pG2pA-Y in the TL-catalyzed cross-linking reaction. A high molar ratio of BAP-Y to Y-pG2pA-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.

Original languageEnglish
Pages (from-to)559-566
Number of pages8
JournalJournal of Bioscience and Bioengineering
Volume126
Issue number5
DOIs
Publication statusPublished - Nov 2018

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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