Site-specific protein cross-linking by peroxidase-catalyzed activation of a tyrosine-containing peptide tag

Kosuke Minamihata, Masahiro Goto, Noriho Kamiya

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62 Citations (Scopus)


Protein modification methods represent fundamental techniques that are applicable in many fields. In this study, a site-specific protein cross-linking based on the oxidative tyrosine coupling reaction was demonstrated. In the presence of horseradish peroxidase (HRP) and H2O2, tyrosine residues undergo one-electron oxidation reactions and form radicals in their phenolic moieties, and these species subsequently react with each other to form dimers or further react to generate polymers. Here, a peptide-tag containing a tyrosine residue(s) (Y-tag, of which the amino acid sequences were either GGGGY or GGYYY) was genetically introduced at the C-terminus of a model protein, Escherichia coli alkaline phosphatase (BAP). Following the incubation of recombinant BAPs with HRP and H2O2, Y-tagged BAPs were efficiently cross-linked with each other, whereas wild-type BAP did not undergo cross-linking, indicating that the tyrosine residues in the Y-tags were recognized by HRP as the substrates. To determine the site-specificity of the cross-linking reaction, the Y-tag was selectively removed by thrombin digestion. The resultant BAP without the Y-tag showed no reactivity in the presence of HRP and H2O2. Conversely, Y-tagged BAPs cross-linked by HRP treatment were almost completely digested into monomeric BAP units following incubation with the protease. Moreover, cross-linked Y-tagged BAPs retained ∼95% of their native enzymatic activity. These results show that HRP catalyzed the site-specific cross-linking of BAPs through tyrosine residues positioned in the C-terminal Y-tag. The site-selective enzymatic oxidative tyrosine coupling reaction should offer a practical option for site-specific and covalent protein modifications.

Original languageEnglish
Pages (from-to)74-81
Number of pages8
JournalBioconjugate Chemistry
Issue number1
Publication statusPublished - Jan 19 2011

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry


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