RESUMO

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.

Assuntos

Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/isolamento & purificação , Xylella/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli , Fator Xa/genética , Fator Xa/metabolismo , Fluorescência , Vetores Genéticos/genética , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência