Construction of Semisynthetic DNA-Protein Conjugates with Phi X174 Gene-A* Protein
Construction of Semisynthetic DNA-Protein Conjugates with Phi X174 Gene-A* Protein
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DOI：
10.1021/bc300118m
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发表时间：
2012-06-01
影响因子：
4.7
通讯作者：
Kobatake, Eiry
中科院分区：
文献类型：
--
作者：
Mashimo, Yasumasa;Maeda, Hitomi;Kobatake, Eiry
DNA蛋白偶联物作为一种多功能的分子工具，在生物技术中被广泛应用，以利用DNA和蛋白质功能的协同效应。随着DNA蛋白偶联物的应用越来越广泛，需要一种易于使用和经济的方法来合成DNA蛋白偶联物。在这项研究中，我们开发了一种通过噬菌体phi X174的基因- a *蛋白（基因- a *）将单链DNA （ssDNA）特异位点标记到重组蛋白（POI）的方法，而不需要对ssDNA进行任何化学修饰。基因a *蛋白是一种位点选择性切割含有基因a *识别序列的寡脱氧核糖核苷酸（ODN）的酶，此时基因a *的酪氨酸残基通过稳定的磷酸酪氨酸连锁键结合到切割位点的5'-磷酸基上。本文构建了三种与基因a *融合的重组蛋白：n端基因a *融合的增强绿色荧光蛋白（EGFP）、c端基因a *融合的EGFP和n端基因a *融合的萤火虫荧光素酶（FLuc）。三种蛋白中由基因a *片段催化的DNA蛋白偶联反应产率达到80-90%，动力学研究表明反应在10 min后达到稳定状态。此外，我们还利用点印迹分析来评估ssDNA与重组基因a *-FLuc蛋白的基因a *片段结合的杂交和适体形成能力。本研究表明，利用重组蛋白与基因a *融合的策略可以提供一个多功能、快速、易于使用和经济的平台来生产DNA蛋白偶联物。
DNA protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA protein conjugates.