(582dj) Protein Polymerization Technique Using Horseradish Peroxidase Catalyzed Tyorsine Coupling Reaction

Cross-linking reaction of proteins offers an enhancement effect of a distinct function of proteins due to the multivalent effect. In addition to that, conjugation of different kinds of proteins creates hybrid biomaterials possessing multiple functions of the building block proteins and such protein conjugates often show great performance caused by the synergistic effect. In this context, we have focused on developing an alternative protein cross-linking technique that enables to generate protein conjugates composed of multiple numbers of proteins. The horseradish peroxidase (HRP) mediated tyrosyl radical coupling reaction was utilized to achieve such protein cross-linking reaction. HRP catalyzes the oxidation reaction of various phenolic compounds by decomposing hydrogen peroxide and generates phenoxy radicals. These radicals then react with each other to form polymers. One of the 20 natural amino acids, tyrosine (Tyr or Y), has phenolic moiety in its side chain. Therefore Tyr can be recognized by HRP and polymerize upon HRP catalysis as well. Our strategy involves the genetic engineering of target proteins to introduce a highly flexible peptide tag containing tyrosine residues (hereafter called Y-tag). We anticipated that HRP recognizes the tyrosine residues in the Y-tag due to steric hindrance and electrostatic interaction between the target proteins and HRP. As model proteins, we selected E.coli alkaline phosphatase and streptavidin. The wild-type of these proteins showed almost no reactivity against HRP treatment, while the Y-tagged proteins showed high reactivity and resulted highly cross-linked protein conjugates. By using this methodology, we have demonstrated protein homoconjugation and protein heteroconjugation and also we very recently reported the control of the Y-tag and HRP mediated proteins conjugation reaction by using electrostatic interactions. In this report, we would like to summarize the recent progress of Y-tag and HRP mediated protein cross-linking techniques.