Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa

in this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37degreesC. with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy. (C) 2004 Elsevier Inc. All rights reserved.37232032

A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle Xray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of DL-glyceraldehyde (K-cat 2.26 s(-1), K-m 8.20 +/- 0.98 mM) and 2-nitrobenzaldehyde (K-cat 11.74 s(-1), K-m 0.14 +/- 0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants. (c) 2006 Elsevier Inc. All rights reserved.453214315

Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa

The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed a-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity. (C) 2003 Elsevier Inc. All rights reserved.33229730

Initial crystallographic studies of a small heat-shock protein from Xylella fastidiosa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The ORF XF2234 in the Xylella fastidiosa genome was identified as encoding a small heat-shock protein of 17.9 kDa (HSP17.9). HSP17.9 was found as one of the proteins that are induced during X. fastidiosa proliferation and infection in citrus culture. Recombinant HSP17.9 was crystallized and surface atomic force microscopy experiments were conducted with the aim of better characterizing the HSP17.9 crystals. X-ray diffraction data were collected at 2.7 angstrom resolution. The crystal belonged to space group P4(3)22, with unit-cell parameters a = 68.90, b = 68.90, c = 72.51 angstrom, and is the first small heat-shock protein to crystallize in this space group.685535539Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES