Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa

被引：3
|
作者：
Paula, DP
Azzoni, AR
Siqueira, SF
Catani, CF
Rosselli, LK
de Souza, AP
机构：
[1] Univ Estadual Campinas, Inst Biol, Dept Genet & Evolut, Genet Engn & Mol Biol, BR-13083970 Campinas, SP, Brazil
[2] Univ Estadual Campinas, Inst Biol, Dept Microbiol & Immunol, Campinas, SP, Brazil
来源：
PROTEIN EXPRESSION AND PURIFICATION | 2003年 / 32卷 / 01期
关键词：
H-NS protein; Xylella fastidiosa; DNA-binding activity or functional genomic;
D O I：
10.1016/S1046-5928(03)00193-1
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
The H-NS protein is one of the major constituents of the nucleoid structure that has been implicated in the DNA packaging and in the global regulation of gene expression. The study of this transcriptional regulator is an effort to fight Xylella fastidiosa, a citrus pathogen responsible for a range of economically important plant diseases, including the citrus variegated chlorosis (CVC). The putative H-NS ORF was cloned into a pET32-Xa/LIC vector in order to overexpress it coupled with fusion tags in Escherichia Coli BL21(DE3). The expressed recombinant protein was purified by immobilized metal affinity chromatography (Ni-NTA resin) and its identity verified by mass spectrometry (MALDI-TOF). Final purification was performed by cation-exchange chromatography (SP Sepharose Fast Flow) and the purified protein was found as a single band on SDS-PAGE. The folding and its DNA binding activity were verified by circular dichroism and fluorescence spectroscopy, respectively. (C) 2003 Elsevier Inc. All rights reserved.
引用
收藏
页码：61 / 67
页数：7
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