title: Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction.
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.
authors: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Japan.
authors: Kosuke
authors: K
authors: Minamihata
authors: Masahiro
authors: M
authors: Goto
authors: Noriho
authors: N
authors: Kamiya
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D001710:Biotin; D019809:Streptavidin; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D013917:Thrombin