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

被引：60

作者：

Sawasaki, Tatsuya ^{[1
,2
]}

Kamura, Nami ^{[1
,2
,3
]}

Matsunaga, Satoko ^{[1
,2
]}

Saeki, Mihoro ^{[3
]}

Tsuchimochi, Masateru ^{[1
,2
]}

Morishita, Ryo ^{[3
]}

Endo, Yaeta ^{[1
,2
,4
]}

机构：

[1] Ehime Univ, Cell Free Sci & Technol Res Ctr, Matsuyama, Ehime 7908577, Japan

[2] Ehime Univ, Venture Business Lab, Matsuyama, Ehime 7908577, Japan

[3] CellFree Sci Co Ltd, Kanagawa 2300046, Japan

[4] RIKEN, Genom Sci Ctr, Yokohama, Kanagawa 2300045, Japan

来源：

FEBS LETTERS | 2008年 / 582卷 / 02期

关键词：

Arabidopsis HY5; DNA-binding tag; cell-free protein synthesis; protein microarray : substrate screening; transcription factor;

D O I：

10.1016/j.febslet.2007.12.004

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

引用

页码：221 / 228

页数：8