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

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 (pG(2)pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG(2)pA conjugate, composed of mainly two to three units of pG(2)pA. 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 pG(2)pA. The affinity of the Y-tagged pG(2)pA 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.