Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate

• Published in: FEBS letters 2008-01, Vol.582 (2), p.221-228
• Main Authors: Sawasaki, Tatsuya, Kamura, Nami, Matsunaga, Satoko, Saeki, Mihoro, Tsuchimochi, Masateru, Morishita, Ryo, Endo, Yaeta
• Format: Article
• Language: English
• Subjects: Arabidopsis; Arabidopsis - physiology; Arabidopsis HY5; Arabidopsis Proteins - physiology; Base Sequence; Basic-Leucine Zipper Transcription Factors - physiology; Cell-free protein synthesis; DNA Primers; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - physiology; DNA-binding tag; Nuclear Proteins - physiology; Plant Proteins - isolation & purification; Plant Proteins - physiology; Protein Array Analysis; Protein microarray: Substrate screening; Sepharose; Short Communication; Short Communications; Transcription factor
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2007.12.004

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.