Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa.
Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST.
Travensolo
Regiane F
RF
Laboratório de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos (USP), São Carlos, Brazil.
Garcia
Wanius
W
Muniz
João R C
JR
Caruso
Célia S
CS
Lemos
Eliana G M
EG
Carrilho
Emanuel
E
Araújo
Ana P U
AP
D002942:Circular Dichroism; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D044167:Xylella / Q000201:enzymology*
D011994:Recombinant Proteins; D005982:Glutathione Transferase