Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants

Autores
Morgenfeld, Mauro Miguel; Vater, Catalina Francisca; Alfano, Edgardo Federico; Boccardo, Noelia Ayelen; Bravo Almonacid, Fernando Felix
Año de publicación
2020
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.
Fil: Morgenfeld, Mauro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Vater, Catalina Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Alfano, Edgardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Boccardo, Noelia Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Materia
HUMAN EPIDERMAL GROWTH FACTOR, EGF
MOLECULAR FARMING
THYLAKOID
TRANSLOCATION
TRANSPLASTOMIC TOBACCO
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:11336/139226
Acceder
id CONICETDig_70d7ba44c3df2bdc505a27f15692f982
oai_identifier_str oai:ri.conicet.gov.ar:11336/139226
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plantsMorgenfeld, Mauro MiguelVater, Catalina FranciscaAlfano, Edgardo FedericoBoccardo, Noelia AyelenBravo Almonacid, Fernando FelixHUMAN EPIDERMAL GROWTH FACTOR, EGFMOLECULAR FARMINGTHYLAKOIDTRANSLOCATIONTRANSPLASTOMIC TOBACCOhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.Fil: Morgenfeld, Mauro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vater, Catalina Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alfano, Edgardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Boccardo, Noelia Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaSpringer2020-06info: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/11336/139226Morgenfeld, Mauro Miguel; Vater, Catalina Francisca; Alfano, Edgardo Federico; Boccardo, Noelia Ayelen; Bravo Almonacid, Fernando Felix; Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants; Springer; Transgenic Research; 29; 3; 6-2020; 295-3050962-8819CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://link.springer.com/10.1007/s11248-020-00199-7info:eu-repo/semantics/altIdentifier/doi/10.1007/s11248-020-00199-7info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:12:30Zoai:ri.conicet.gov.ar:11336/139226instacron: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 10:12:30.648CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
title Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
spellingShingle Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
Morgenfeld, Mauro Miguel
HUMAN EPIDERMAL GROWTH FACTOR, EGF
MOLECULAR FARMING
THYLAKOID
TRANSLOCATION
TRANSPLASTOMIC TOBACCO
title_short Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
title_full Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
title_fullStr Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
title_full_unstemmed Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
title_sort Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants
dc.creator.none.fl_str_mv Morgenfeld, Mauro Miguel
Vater, Catalina Francisca
Alfano, Edgardo Federico
Boccardo, Noelia Ayelen
Bravo Almonacid, Fernando Felix
author Morgenfeld, Mauro Miguel
author_facet Morgenfeld, Mauro Miguel
Vater, Catalina Francisca
Alfano, Edgardo Federico
Boccardo, Noelia Ayelen
Bravo Almonacid, Fernando Felix
author_role author
author2 Vater, Catalina Francisca
Alfano, Edgardo Federico
Boccardo, Noelia Ayelen
Bravo Almonacid, Fernando Felix
author2_role author
author
author
author
dc.subject.none.fl_str_mv HUMAN EPIDERMAL GROWTH FACTOR, EGF
MOLECULAR FARMING
THYLAKOID
TRANSLOCATION
TRANSPLASTOMIC TOBACCO
topic HUMAN EPIDERMAL GROWTH FACTOR, EGF
MOLECULAR FARMING
THYLAKOID
TRANSLOCATION
TRANSPLASTOMIC TOBACCO
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.
Fil: Morgenfeld, Mauro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Vater, Catalina Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Alfano, Edgardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Boccardo, Noelia Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
description Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.
publishDate 2020
dc.date.none.fl_str_mv 2020-06
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/11336/139226
Morgenfeld, Mauro Miguel; Vater, Catalina Francisca; Alfano, Edgardo Federico; Boccardo, Noelia Ayelen; Bravo Almonacid, Fernando Felix; Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants; Springer; Transgenic Research; 29; 3; 6-2020; 295-305
0962-8819
CONICET Digital
CONICET
url http://hdl.handle.net/11336/139226
identifier_str_mv Morgenfeld, Mauro Miguel; Vater, Catalina Francisca; Alfano, Edgardo Federico; Boccardo, Noelia Ayelen; Bravo Almonacid, Fernando Felix; Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants; Springer; Transgenic Research; 29; 3; 6-2020; 295-305
0962-8819
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://link.springer.com/10.1007/s11248-020-00199-7
info:eu-repo/semantics/altIdentifier/doi/10.1007/s11248-020-00199-7
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 Springer
publisher.none.fl_str_mv Springer
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
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score 13.070432

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