High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit

Autores
Gomez, Rodrigo Lionel; Galles, Celina; Spampinato, Claudia Patricia
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His6-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.
Fil: Gomez, Rodrigo Lionel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Fil: Galles, Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Fil: Spampinato, Claudia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Materia
Msh2
Heterologous Expression
E. Coli
Polyclonal Antibody
Msh2
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/15425
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/15425
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunitGomez, Rodrigo LionelGalles, CelinaSpampinato, Claudia PatriciaMsh2Heterologous ExpressionE. ColiPolyclonal AntibodyMsh2https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His6-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.Fil: Gomez, Rodrigo Lionel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Galles, Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Spampinato, Claudia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaHumana Press2011-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/15425Gomez, Rodrigo Lionel; Galles, Celina; Spampinato, Claudia Patricia; High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit; Humana Press; Molecular Biotechnology; 47; 2; 2-2011; 120-1291073-60851559-0305enginfo:eu-repo/semantics/altIdentifier/doi/10.1007/s12033-010-9319-9info:eu-repo/semantics/altIdentifier/url/https://link.springer.com/article/10.1007%2Fs12033-010-9319-9info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:55:02Zoai:ri.conicet.gov.ar:11336/15425instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:55:02.53CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
title High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
spellingShingle High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
Gomez, Rodrigo Lionel
Msh2
Heterologous Expression
E. Coli
Polyclonal Antibody
Msh2
title_short High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
title_full High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
title_fullStr High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
title_full_unstemmed High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
title_sort High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit
dc.creator.none.fl_str_mv Gomez, Rodrigo Lionel
Galles, Celina
Spampinato, Claudia Patricia
author Gomez, Rodrigo Lionel
author_facet Gomez, Rodrigo Lionel
Galles, Celina
Spampinato, Claudia Patricia
author_role author
author2 Galles, Celina
Spampinato, Claudia Patricia
author2_role author
author
dc.subject.none.fl_str_mv Msh2
Heterologous Expression
E. Coli
Polyclonal Antibody
Msh2
topic Msh2
Heterologous Expression
E. Coli
Polyclonal Antibody
Msh2
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His6-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.
Fil: Gomez, Rodrigo Lionel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Fil: Galles, Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Fil: Spampinato, Claudia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
description Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His6-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.
publishDate 2011
dc.date.none.fl_str_mv 2011-02
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/15425
Gomez, Rodrigo Lionel; Galles, Celina; Spampinato, Claudia Patricia; High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit; Humana Press; Molecular Biotechnology; 47; 2; 2-2011; 120-129
1073-6085
1559-0305
url http://hdl.handle.net/11336/15425
identifier_str_mv Gomez, Rodrigo Lionel; Galles, Celina; Spampinato, Claudia Patricia; High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit; Humana Press; Molecular Biotechnology; 47; 2; 2-2011; 120-129
1073-6085
1559-0305
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1007/s12033-010-9319-9
info:eu-repo/semantics/altIdentifier/url/https://link.springer.com/article/10.1007%2Fs12033-010-9319-9
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Humana Press
publisher.none.fl_str_mv Humana Press
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1844613661459480576
score 13.070432

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