Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG

Kosuke Minamihata, Masahiro Goto, Noriho Kamiya

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5 Citations (Scopus)

Abstract

Cross-linking proteins offers an approach to enhance the distinct function of proteins due to the multivalent effect. In this study, we demonstrated the preparation of a multivalent antibody-binding protein possessing high affinity to IgG by conjugating a number of antibody-binding proteins using the horseradish peroxidase (HRP)-mediated protein conjugation method. By introducing a peptide tag containing a tyrosine (Y-tag) to the C-terminus of the model protein, a chimera protein of protein G and protein A (pG2pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG2pA conjugate, composed of mainly two to three units of pG2pA. The cross-linking occurred site specifically at the Tyr residue in the Y-tag and introduction of the Y-tag showed no effect on the function of pG2pA. The affinity of the Y-tagged pG2pA conjugate against IgG clearly increased because of the multivalent effect, demonstrating the benefit of this protein cross-linking reaction, which yields functional protein oligomers. Such multivalent protein conjugates created by this reaction should have potential to be used in ELISA and Western blotting applications in which highly sensitive detection of target molecules is desired.

Original languageEnglish
Pages (from-to)222-226
Number of pages5
JournalBiotechnology Journal
Volume10
Issue number1
DOIs
Publication statusPublished - Jan 1 2015

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Horseradish Peroxidase
Carrier Proteins
Immunoglobulin G
Antibodies
Proteins
Cross Reactions
Staphylococcal Protein A
GTP-Binding Proteins
Tyrosine
Western Blotting
Enzyme-Linked Immunosorbent Assay
Peptides

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

Cite this

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