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

Azzoni, Adriano Rodrigues; Tada, Susely F.S.; Rosselli, Luciana Kauer; Paula, Danielli Parrilha de; Catani, Cleide Ferreira; Sabino, Adão A.; Barbosa, J.A.R.G.; Guimarães, B.G.; Eberlin, Marcos N.; Medrano, Francisco J.; Souza, Anete Pereira de

doi:10.1016/j.pep.2003.10.007

Cited by 13 publications

(11 citation statements)

References 12 publications

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“…We demonstrated chaperone activity for S.so-HSP20 of S. solfataricus P2 at physiological and heat shock temperatures. The ability of S.so-HSP20 to protect against heat-induced aggregation of citrate synthase (Keisuke et al 2001; Ding et al 2008; Martin et al 2008; Angelo et al 2004) and insulin B chain (Azzoni et al 2004; Satoru 2006) indicates that S.so-HSP20 possesses chaperone activity and the ability to recognize and bind unfolded proteins to prevent their aggregation in vitro. The relationship between cellular thermotolerance and S.so-HSP20 expression in an E. coli expression system has not previously been reported.…”

Section: Discussionmentioning

confidence: 99%

Thermotolerance and molecular chaperone function of the small heat shock protein HSP20 from hyperthermophilic archaeon, Sulfolobus solfataricus P2

Yang

Lü

et al. 2012

Cell Stress and Chaperones

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Small heat shock proteins are ubiquitous in all three domains (Archaea, Bacteria and Eukarya) and possess molecular chaperone activity by binding to unfolded polypeptides and preventing aggregation of proteins in vitro. The functions of a small heat shock protein (S.so-HSP20) from the hyperthermophilic archaeon, Sulfolobus solfataricus P2 have not been described. In the present study, we used real-time polymerase chain reaction analysis to measure mRNA expression of S.so-HSP20 in S. solfataricus P2 and found that it was induced by temperatures that were substantially lower (60°C) or higher (80°C) than the optimal temperature for S. solfataricus P2 (75°C). The expression of S.so-HSP20 mRNA was also up-regulated by cold shock (4°C). Escherichia coli cells expressing S.so-HSP20 showed greater thermotolerance in response to temperature shock (50°C, 4°C). By assaying enzyme activities, S.so-HSP20 was found to promote the proper folding of thermo-denatured citrate synthase and insulin B chain. These results suggest that S.so-HSP20 promotes thermotolerance and engages in chaperone-like activity during the stress response.

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Section: Discussionmentioning

confidence: 99%

Thermotolerance and molecular chaperone function of the small heat shock protein HSP20 from hyperthermophilic archaeon, Sulfolobus solfataricus P2

Yang

Lü

et al. 2012

Cell Stress and Chaperones

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“…Crystallization. Protein cloning, expression and purification were conducted as described by Azzoni et al (2004). Briefly, the full-length HSP17.9 gene (UniProtKB Q9PBB0) was subcloned into pET32-Xa/LIC (Novagen) and gene integrity was confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…A clear contrast among the tested point groups, with a BALBES Q-factor parameter of 0.834 for the template model and a probability of 99% for the solution obtained, further supports its correctness. It is interesting to note that although HSP17.9 exhibits a tendency to form oligomeric complexes (Azzoni et al, 2004), in its function as a chaperone, a single monomer was found in the crystal asymmetric unit. Most probably, as in the case of other smHSPs, monodisperse oligomers might be assembled from dimers in solution (Takeda et al, 2011;Bagné ris et al, 2009;Stamler et al, 2005), thus providing a necessary condition for crystals to grow.…”

Section: Diffraction Data Collection and Processingmentioning

confidence: 99%

“…The smHSP from Xylella fastidiosa, the infectious agent of citrus variegated chlorosis (CVC), was identified after genomic sequencing of the phytopathogen by the Brazilian Consortium ONSA (Simpson et al, 2000). HSP17.9 is the gene product of ORF XF2234 in the X. fastidiosa 9a5c genome, which results in a protein of 17.9 kDa (Azzoni et al, 2004). Citrus culture plays an important role in the Brazilian economy and infection caused by X. fastidiosa is responsible for a loss of approximately US $280-320 million per year (http:// www.fundecitrus.com.br/).…”

Section: Introductionmentioning

confidence: 99%

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Initial crystallographic studies of a small heat-shock protein fromXylella fastidiosa

et al. 2012

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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 Å resolution. The crystal belonged to space group P4 3 22, with unit-cell parameters a = 68.90, b = 68.90, c = 72.51 Å , and is the first small heat-shock protein to crystallize in this space group.

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“…DTT is known to attack the disulphide bond in proteins causing unfolding with the loss of tertiary structure (Azzoni et al, 2004). For these experiments, insulin was used as the analyte protein.…”

Section: Investigating the Effect Of Chemical Stressmentioning

confidence: 99%

Detecting and exploring partially unfolded states of proteins using a sensor with chaperone bound to its surface

George

Bilek

McKenzie

2008

Biosensors and Bioelectronics

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

Cited by 13 publications

References 12 publications

Thermotolerance and molecular chaperone function of the small heat shock protein HSP20 from hyperthermophilic archaeon, Sulfolobus solfataricus P2

Thermotolerance and molecular chaperone function of the small heat shock protein HSP20 from hyperthermophilic archaeon, Sulfolobus solfataricus P2

Initial crystallographic studies of a small heat-shock protein fromXylella fastidiosa

Detecting and exploring partially unfolded states of proteins using a sensor with chaperone bound to its surface

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