Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction

Kosuke Minamihata, Masahiro Goto, Noriho Kamiya

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Combining different proteins can integrate the functions of each protein to produce novel protein conjugates with wider ranges of applications. We have previously introduced a peptide containing tyrosine residues (Y-tag) at the C-terminus of Escherichia coli alkaline phosphatase (BAP). The tyrosine residues in the Y-tag were efficiently recognized by horseradish peroxidase (HRP) and were site-specifically cross-linked with each other to yield BAP homoconjugates. In this study, the HRP-catalyzed tyrosine coupling reaction was used for protein heteroconjugation. Streptavidin (SA) was selected as the conjugation partner for BAP. The Y-tag (GGGGY) was genetically introduced to the C-terminus of SA. Prior to heteroconjugation, the reactivity of the Y-tagged SA was examined. The Y-tagged SA cross-linked to form an SA homoconjugate upon HRP treatment, whereas wild-type SA remained essentially intact. In the heteroconjugation reaction of BAP and SA, the Y-tagged BAP and SA were efficiently cross-linked with each other upon HRP treatment. The functions of the BAP-SA conjugates were evaluated by measuring the BAP enzymatic activity on a biotin-coated plate. The BAP-SA conjugate tethered to the plate showed BAP enzymatic activity, indicating that both BAP and SA retained their functions following heteroconjugation. The BAP-SA conjugate prepared from both Y-tagged BAP and SA showed the highest enzymatic activity on the biotin-coated plates. This result illustrates the advantage of the protein conjugation reaction in which multiple numbers of proteins can be conjugated at the same time.

Original languageEnglish
Pages (from-to)2332-2338
Number of pages7
JournalBioconjugate Chemistry
Volume22
Issue number11
DOIs
Publication statusPublished - Nov 16 2011

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Streptavidin
Peroxidase
Tyrosine
Proteins
Horseradish Peroxidase
Phosphatases
Biotin
Escherichia coli
Peptides
Alkaline Phosphatase

All Science Journal Classification (ASJC) codes

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

Cite this

Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction. / Minamihata, Kosuke; Goto, Masahiro; Kamiya, Noriho.

In: Bioconjugate Chemistry, Vol. 22, No. 11, 16.11.2011, p. 2332-2338.

Research output: Contribution to journalArticle

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