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 (pG2 pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG2 pA conjugate, composed of mainly two to three units of pG2 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 pG2 pA. The affinity of the Y-tagged pG2 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.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.
Kosuke
K
Minamihata
Masahiro
M
Goto
Noriho
N
Kamiya
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D002645:Chickens; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000737:chemistry
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014443:Tyrosine; D006735:Horseradish Peroxidase
Antibody binding protein; Conjugation; Horseradish peroxidase; IgG cross-linking; Tyrosine coupling reaction