Protein Heteroconjugation by the Peroxidase-Catalyzed Tyrosine Coupling Reaction
Protein Heteroconjugation by the Peroxidase-Catalyzed Tyrosine Coupling Reaction
复制标题
DOI：
10.1021/bc200420v
复制
发表时间：
2011-11-01
影响因子：
4.7
通讯作者：
Kamiya, Noriho
中科院分区：
文献类型：
--
作者：
Minamihata, Kosuke;Goto, Masahiro;Kamiya, Noriho
结合不同的蛋白质可以整合每种蛋白质的功能，产生应用范围更广的新型蛋白质结合物。我们之前已经在大肠杆菌碱性磷酸酶(BAP)的C末端引入了一个含有酪氨酸残基的多肽(Y-Tag)。辣根过氧化物酶(HRP)能有效识别Y-TAG中的酪氨酸残基，并相互定点交联，形成BAP同源偶联物。本研究利用HRP催化的酪氨酸偶联反应进行蛋白质杂化偶联。链霉亲和素(SA)被选为BAP的偶联伙伴。Y标记(GGGGY)被遗传导入SA的C-末端。在杂合之前，检测了Y标记SA的反应性。经HRP处理后，Y标记的SA发生交叉连接，形成SA同源结合物，而野生型SA基本保持不变。在BAP和SA的杂合反应中，Y标记的BAP和SA在FIR？治疗。通过在生物素包被的平板上测定BAP酶活性来评价BAP-SA偶联物的功能。BAP-SA偶联物具有BAP酶活性，说明BAP和SA在异源偶联后都保留了各自的功能。由Y标记的BAP和SA制备的BAP-SA结合物在生物素包被的平板上显示出最高的酶活性。这一结果说明了蛋白质偶联反应的优势，在该反应中可以同时偶联多个蛋白质。
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 FIR? 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.