Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

Autores
Coragliotti, Anna T.; Beligni, María Verónica; Franklin, Scott E.; Mayfield, Stephen P.
Año de publicación
2010
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions could this be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.
Fil: Coragliotti, Anna T.. No especifíca;
Fil: Beligni, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina
Fil: Franklin, Scott E.. No especifíca;
Fil: Mayfield, Stephen P.. University of California at San Diego; Estados Unidos
Materia
Chlamydomonas
Biotechnology of microalgae
Chloroplast translation
Recombinant protein expression
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/245605
Acceder
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network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii ChloroplastCoragliotti, Anna T.Beligni, María VerónicaFranklin, Scott E.Mayfield, Stephen P.ChlamydomonasBiotechnology of microalgaeChloroplast translationRecombinant protein expressionhttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions could this be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.Fil: Coragliotti, Anna T.. No especifíca;Fil: Beligni, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Franklin, Scott E.. No especifíca;Fil: Mayfield, Stephen P.. University of California at San Diego; Estados UnidosHumana Press2010-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/245605Coragliotti, Anna T.; Beligni, María Verónica; Franklin, Scott E.; Mayfield, Stephen P.; Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast; Humana Press; Molecular Biotechnology; 48; 1; 11-2010; 60-751073-6085CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://link.springer.com/article/10.1007/s12033-010-9348-4info:eu-repo/semantics/altIdentifier/doi/10.1007/s12033-010-9348-4info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:44:23Zoai:ri.conicet.gov.ar:11336/245605instacron: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:34982025-09-03 09:44:23.798CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
title Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
spellingShingle Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
Coragliotti, Anna T.
Chlamydomonas
Biotechnology of microalgae
Chloroplast translation
Recombinant protein expression
title_short Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
title_full Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
title_fullStr Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
title_full_unstemmed Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
title_sort Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
dc.creator.none.fl_str_mv Coragliotti, Anna T.
Beligni, María Verónica
Franklin, Scott E.
Mayfield, Stephen P.
author Coragliotti, Anna T.
author_facet Coragliotti, Anna T.
Beligni, María Verónica
Franklin, Scott E.
Mayfield, Stephen P.
author_role author
author2 Beligni, María Verónica
Franklin, Scott E.
Mayfield, Stephen P.
author2_role author
author
author
dc.subject.none.fl_str_mv Chlamydomonas
Biotechnology of microalgae
Chloroplast translation
Recombinant protein expression
topic Chlamydomonas
Biotechnology of microalgae
Chloroplast translation
Recombinant protein expression
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions could this be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.
Fil: Coragliotti, Anna T.. No especifíca;
Fil: Beligni, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina
Fil: Franklin, Scott E.. No especifíca;
Fil: Mayfield, Stephen P.. University of California at San Diego; Estados Unidos
description In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions could this be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.
publishDate 2010
dc.date.none.fl_str_mv 2010-11
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/11336/245605
Coragliotti, Anna T.; Beligni, María Verónica; Franklin, Scott E.; Mayfield, Stephen P.; Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast; Humana Press; Molecular Biotechnology; 48; 1; 11-2010; 60-75
1073-6085
CONICET Digital
CONICET
url http://hdl.handle.net/11336/245605
identifier_str_mv Coragliotti, Anna T.; Beligni, María Verónica; Franklin, Scott E.; Mayfield, Stephen P.; Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast; Humana Press; Molecular Biotechnology; 48; 1; 11-2010; 60-75
1073-6085
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://link.springer.com/article/10.1007/s12033-010-9348-4
info:eu-repo/semantics/altIdentifier/doi/10.1007/s12033-010-9348-4
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc/2.5/ar/
dc.format.none.fl_str_mv 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
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score 13.13397

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