Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli
- Autores
- Santos, Javier; Antón, Erica A.; Marino Buslje, Cristina; Valdez, Silvina Noemi; Villanueva, Ana L.; Sica, Mauricio Pablo; Iacono, Ruben Francisco; Maffia, Paulo Cesar; Poskus, Edgardo; Ermacora, Mario Roberto
- Año de publicación
- 2000
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.
Fil: Santos, Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Antón, Erica A.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Marino Buslje, Cristina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: Villanueva, Ana L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: Sica, Mauricio Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: Maffia, Paulo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina - Materia
-
Gad
Threonin
Recombinant
Thiorredoxin - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/41519
Ver los metadatos del registro completo
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Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coliSantos, JavierAntón, Erica A.Marino Buslje, CristinaValdez, Silvina NoemiVillanueva, Ana L.Sica, Mauricio PabloIacono, Ruben FranciscoMaffia, Paulo CesarPoskus, EdgardoErmacora, Mario RobertoGadThreoninRecombinantThiorredoxinhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.Fil: Santos, Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Antón, Erica A.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Marino Buslje, Cristina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Villanueva, Ana L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sica, Mauricio Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Maffia, Paulo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaPortland Press2000-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/41519Santos, Javier; Antón, Erica A.; Marino Buslje, Cristina; Valdez, Silvina Noemi; Villanueva, Ana L.; et al.; Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli; Portland Press; Biotechnology and Applied Biochemistry; 31; 3; 6-2000; 205-2120885-4513CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://iubmb.onlinelibrary.wiley.com/doi/10.1042/BA19990103info:eu-repo/semantics/altIdentifier/doi/10.1042/BA19990103info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:26:46Zoai:ri.conicet.gov.ar:11336/41519instacron: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 10:26:46.855CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
title |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
spellingShingle |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli Santos, Javier Gad Threonin Recombinant Thiorredoxin |
title_short |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
title_full |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
title_fullStr |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
title_full_unstemmed |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
title_sort |
Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli |
dc.creator.none.fl_str_mv |
Santos, Javier Antón, Erica A. Marino Buslje, Cristina Valdez, Silvina Noemi Villanueva, Ana L. Sica, Mauricio Pablo Iacono, Ruben Francisco Maffia, Paulo Cesar Poskus, Edgardo Ermacora, Mario Roberto |
author |
Santos, Javier |
author_facet |
Santos, Javier Antón, Erica A. Marino Buslje, Cristina Valdez, Silvina Noemi Villanueva, Ana L. Sica, Mauricio Pablo Iacono, Ruben Francisco Maffia, Paulo Cesar Poskus, Edgardo Ermacora, Mario Roberto |
author_role |
author |
author2 |
Antón, Erica A. Marino Buslje, Cristina Valdez, Silvina Noemi Villanueva, Ana L. Sica, Mauricio Pablo Iacono, Ruben Francisco Maffia, Paulo Cesar Poskus, Edgardo Ermacora, Mario Roberto |
author2_role |
author author author author author author author author author |
dc.subject.none.fl_str_mv |
Gad Threonin Recombinant Thiorredoxin |
topic |
Gad Threonin Recombinant Thiorredoxin |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/3.4 https://purl.org/becyt/ford/3 |
dc.description.none.fl_txt_mv |
Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65. Fil: Santos, Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Antón, Erica A.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Marino Buslje, Cristina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Villanueva, Ana L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Sica, Mauricio Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Maffia, Paulo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina |
description |
Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65. |
publishDate |
2000 |
dc.date.none.fl_str_mv |
2000-06 |
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/11336/41519 Santos, Javier; Antón, Erica A.; Marino Buslje, Cristina; Valdez, Silvina Noemi; Villanueva, Ana L.; et al.; Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli; Portland Press; Biotechnology and Applied Biochemistry; 31; 3; 6-2000; 205-212 0885-4513 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/41519 |
identifier_str_mv |
Santos, Javier; Antón, Erica A.; Marino Buslje, Cristina; Valdez, Silvina Noemi; Villanueva, Ana L.; et al.; Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli; Portland Press; Biotechnology and Applied Biochemistry; 31; 3; 6-2000; 205-212 0885-4513 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/https://iubmb.onlinelibrary.wiley.com/doi/10.1042/BA19990103 info:eu-repo/semantics/altIdentifier/doi/10.1042/BA19990103 |
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 application/pdf |
dc.publisher.none.fl_str_mv |
Portland Press |
publisher.none.fl_str_mv |
Portland Press |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) |
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CONICET Digital (CONICET) |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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1844614269125001216 |
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13.070432 |