A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri

Detalhes bibliográficos
Autor(a) principal: Dantas, Giordanni C. [UNESP]
Data de Publicação: 2016
Outros Autores: Martins, Paula M. M. [UNESP], Martins, Daniela A. B. [UNESP], Gomes, Eleni [UNESP], Ferreira, Henrique [UNESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.1016/j.bjm.2016.01.026
http://hdl.handle.net/11449/161506
Resumo: Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that similar to 37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.
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spelling A protein expression system for tandem affinity purification in Xanthomonas citri subsp citriCitrus cankerExpression vectorsTAP-tagCitrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that similar to 37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Estadual Paulista, Inst Biociencias, Dept Bioquim & Microbiol, Rio Claro, SP, BrazilUniv Estadual Paulista, Inst Quim, Dept Bioquim & Tecnol Quim, Araraquara, SP, BrazilUniv Estadual Paulista, Dept Biol, Sao Jose Do Rio Preto, SP, BrazilUniv Estadual Paulista, Inst Biociencias, Dept Bioquim & Microbiol, Rio Claro, SP, BrazilUniv Estadual Paulista, Inst Quim, Dept Bioquim & Tecnol Quim, Araraquara, SP, BrazilUniv Estadual Paulista, Dept Biol, Sao Jose Do Rio Preto, SP, BrazilFAPESP: FAPESP 2013/19806-5FAPESP: 2006/59494-9FAPESP: 2004/09173-6FAPESP: 2013/50367-8Soc Brasileira MicrobiologiaUniversidade Estadual Paulista (Unesp)Dantas, Giordanni C. [UNESP]Martins, Paula M. M. [UNESP]Martins, Daniela A. B. [UNESP]Gomes, Eleni [UNESP]Ferreira, Henrique [UNESP]2018-11-26T16:33:00Z2018-11-26T16:33:00Z2016-04-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article518-526application/pdfhttp://dx.doi.org/10.1016/j.bjm.2016.01.026Brazilian Journal Of Microbiology. Sao Paulo: Soc Brasileira Microbiologia, v. 47, n. 2, p. 518-526, 2016.1517-8382http://hdl.handle.net/11449/16150610.1016/j.bjm.2016.01.026S1517-83822016000200518WOS:000376016600034S1517-83822016000200518.pdfWeb of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengBrazilian Journal Of Microbiology0,630info:eu-repo/semantics/openAccess2023-12-09T06:24:35Zoai:repositorio.unesp.br:11449/161506Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T19:53:02.157571Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
title A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
spellingShingle A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
Dantas, Giordanni C. [UNESP]
Citrus canker
Expression vectors
TAP-tag
title_short A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
title_full A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
title_fullStr A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
title_full_unstemmed A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
title_sort A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
author Dantas, Giordanni C. [UNESP]
author_facet Dantas, Giordanni C. [UNESP]
Martins, Paula M. M. [UNESP]
Martins, Daniela A. B. [UNESP]
Gomes, Eleni [UNESP]
Ferreira, Henrique [UNESP]
author_role author
author2 Martins, Paula M. M. [UNESP]
Martins, Daniela A. B. [UNESP]
Gomes, Eleni [UNESP]
Ferreira, Henrique [UNESP]
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual Paulista (Unesp)
dc.contributor.author.fl_str_mv Dantas, Giordanni C. [UNESP]
Martins, Paula M. M. [UNESP]
Martins, Daniela A. B. [UNESP]
Gomes, Eleni [UNESP]
Ferreira, Henrique [UNESP]
dc.subject.por.fl_str_mv Citrus canker
Expression vectors
TAP-tag
topic Citrus canker
Expression vectors
TAP-tag
description Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that similar to 37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.
publishDate 2016
dc.date.none.fl_str_mv 2016-04-01
2018-11-26T16:33:00Z
2018-11-26T16:33:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.1016/j.bjm.2016.01.026
Brazilian Journal Of Microbiology. Sao Paulo: Soc Brasileira Microbiologia, v. 47, n. 2, p. 518-526, 2016.
1517-8382
http://hdl.handle.net/11449/161506
10.1016/j.bjm.2016.01.026
S1517-83822016000200518
WOS:000376016600034
S1517-83822016000200518.pdf
url http://dx.doi.org/10.1016/j.bjm.2016.01.026
http://hdl.handle.net/11449/161506
identifier_str_mv Brazilian Journal Of Microbiology. Sao Paulo: Soc Brasileira Microbiologia, v. 47, n. 2, p. 518-526, 2016.
1517-8382
10.1016/j.bjm.2016.01.026
S1517-83822016000200518
WOS:000376016600034
S1517-83822016000200518.pdf
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Brazilian Journal Of Microbiology
0,630
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 518-526
application/pdf
dc.publisher.none.fl_str_mv Soc Brasileira Microbiologia
publisher.none.fl_str_mv Soc Brasileira Microbiologia
dc.source.none.fl_str_mv Web of Science
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv
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