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首页> 外文期刊>Journal of Molecular Biology >Searching for DNA-protein Interactions by Lambda Phage Display.
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Searching for DNA-protein Interactions by Lambda Phage Display.

机译：通过Lambda Phage Display搜索DNA-蛋白质相互作用。

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摘要

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.
机译：我们将噬菌体展示技术应用于DNA-蛋白质相互作用研究。从高度分化的MMH E14鼠肝细胞系中产生了噬菌体λ表面上显示的cDNA表达文库。使用富含肝脏的转录因子HNF1alpha基因的启动子序列作为连接物来选择该文库，可确定与该调控序列不同区域特异性相互作用的DNA结合结构域。所选噬菌体之一显示出与分化特异性元件结合蛋白，血管活性肠肽受体阻遏蛋白和复制因子C共有的DNA结合结构域具有100％的同一性，并进行了进一步研究。在不依赖噬菌体的情况下，证实了所选蛋白结构域的特异性结合。通过使用选定的噬菌体和与编码的DNA结合结构域融合的细菌表达的谷胱甘肽S-转移酶蛋白结合ELISA和South-Western分析，鉴定出了多个共有共同共有基序的相邻DNA结合位点。所描述的策略代表了一种识别与DNA调控元件结合的蛋白质的强大工具。

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