Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate.
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.
Sawasaki
Tatsuya
T
Cell-Free Science and Technology Research Center, and The Venture Business Laboratory, Ehime University, Matsuyama, Ehime, Japan. sawasaki@eng.ehime-u.ac.jp
Kamura
Nami
N
Matsunaga
Satoko
S
Saeki
Mihoro
M
Tsuchimochi
Masateru
M
Morishita
Ryo
R
Endo
Yaeta
Y
D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000502:physiology*; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000502:physiology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D009687:Nuclear Proteins / Q000502:physiology*; D010940:Plant Proteins / Q000302:isolation & purification / Q000502:physiology*; D040081:Protein Array Analysis; D012685:Sepharose
D029681:Arabidopsis Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D017931:DNA Primers; D004268:DNA-Binding Proteins; C109254:HY5 protein, Arabidopsis; D009687:Nuclear Proteins; D010940:Plant Proteins; D012685:Sepharose