拟南芥HY5蛋白作为一种DNA结合标签，用于在琼脂糖/DNA微孔板上纯化蛋白质并将其功能固定化。
Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate.
作者信息
Sawasaki Tatsuya, Kamura Nami, Matsunaga Satoko, Saeki Mihoro, Tsuchimochi Masateru, Morishita Ryo, Endo Yaeta
机构信息
Cell-Free Science and Technology Research Center, and The Venture Business Laboratory, Ehime University, Matsuyama, Ehime, Japan.
出版信息
FEBS Lett. 2008 Jan 23;582(2):221-8. doi: 10.1016/j.febslet.2007.12.004. Epub 2007 Dec 17.
Abstract
Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.
摘要
蛋白质微阵列被认为是高通量蛋白质功能分析的关键分析工具之一。在此，我们报道拟南芥HY5作为一种新型的蛋白质DNA结合标签（DBtag）。我们还证明了带有DBtag的蛋白质可以固定在新设计的琼脂糖/DNA微孔板上并进行纯化。此外，我们展示了该微阵列的三种应用：（1）检测带有DBtag的人类蛋白激酶的自磷酸化活性以及星形孢菌素对其活性的抑制，（2）病毒蛋白酶和半胱天冬酶3对带有DBtag的蛋白质的特异性切割，以及（3）检测带有DBtag的UBE2N和UBE2v1之间的蛋白质-蛋白质相互作用。因此，该方法可能有助于对多种蛋白质进行快速功能分析。
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