A protein expression system for tandem affinity purification in Xanthomonas citri subsp citri
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Outros Autores: | , , , |
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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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 |
|
_version_ |
1808129134172307456 |