Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa.
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.
Paula
Débora Pires
DP
Genetic Engineering and Molecular Biology, Center, Department of Genetic and Evolution, Institute of Biology, The State University of Campinas, CP 6010, 13083970 Campinas, SP, Brazil.
Azzoni
Adriano Rodrigues
AR
Siqueira
Susely Ferraz
SF
Catani
Cleide Ferreira
CF
Rosselli
Luciana Kauer
LK
de Souza
Anete Pereira
AP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D005822:Genetic Vectors / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D044167:Xylella / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D011993:Recombinant Fusion Proteins; D015951:Factor Xa