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首页> 外文期刊>Journal of Biotechnology >Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface
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Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface

机译：将DNA结合蛋白直接固定在同源DNA修饰的芯片表面上

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摘要

Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(l-lysine) for sequence-specific DNA-protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein-protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.
机译：在这里，我们描述了一种有用的方法，用于将具有DNA结合结构域（DNA-BD）的蛋白质定点固定在相关的DNA涂层的金表面上，用于表面等离振子共振（SPR）成像分析。为了评估该程序的性能，我们利用了两个DNA-BD，酵母GAL4 DNA-BD和细菌LexA DNA-BD。在将同源双链DNA（dsDNA）固定到具有单层聚（1-赖氨酸）的金芯片表面以进行序列特异性DNA-蛋白质相互作用后，纯化重组GAL4 DNA-BD：EGFP和LexA DNA-BD ：RFP融合蛋白应用于dsDNA斑点金芯片，随后使用SPR成像系统进行分析。因此，重组DNA结合蛋白GAL4 DNA-BD：EGFP和LexA DNA-BD：RFP被证明与金芯片上的同源DNA序列选择性结合。总的来说，我们的研究结果表明，序列特异性dsDNA微阵列方法可用于以平行形式将DNA结合蛋白定点固定在金薄膜上，从而有可能用于转录因子结合谱分析。以及监测具有DNA结合结构域作为融合标签的靶蛋白与其结合伴侣之间的蛋白-蛋白相互作用。

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