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

Kosuke Minamihata, Masahiro Goto, Noriho Kamiya

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

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
Volume22
Issue number1
DOIs
Publication statusPublished - Jan 19 2011

Fingerprint

Horseradish Peroxidase
Peptides
Peroxidase
Tyrosine
Chemical activation
Proteins
Oxidative Coupling
Phosphatases
Dimers
Escherichia coli
Amino acids
Escherichia coli Proteins
Cross Reactions
Thrombin
Oxidation
Alkaline Phosphatase
Electrons
Digestion
Amino Acid Sequence
Polymers

All Science Journal Classification (ASJC) codes

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

Cite this

Site-specific protein cross-linking by peroxidase-catalyzed activation of a tyrosine-containing peptide tag. / Minamihata, Kosuke; Goto, Masahiro; Kamiya, Noriho.

In: Bioconjugate Chemistry, Vol. 22, No. 1, 19.01.2011, p. 74-81.

Research output: Contribution to journalArticle

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