Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants

Autores
González, Pablo A.; Puccio, Franco Damián; Zelada, Alicia Mercedes
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The development of alternative subunit based-vaccines against tuberculosis is necessary due to variable efficiency and some security concerns of the BCG vaccine. The aim of this work was evaluate the production of the Mycobacterium tuberculosis Ag85B antigen fused to Potato Virus X Coat Protein (PVX-CP) by transient expression in Nicotiana benthamiana for subunit-based tuberculosis vaccine formulation. A codon-optimized M. tuberculosis Ag85B gene was fused to PVX-CP and expressed both as a full length precursor and as a mature version lacking the leader peptide. Signal peptides of N. tabacum genes were added to precursor and mature Ag85B-CP to compare the efficiency of cytoplasmic and apoplastic expression. Constructs were agroinfiltrated into N. benthamiana leaves and the yield and integrity of recombinant proteins were analysed. Glycosylation status was determined by treatment with peptide N-glycosidase F. The highest amounts of fusion protein were obtained by expressing mature Ag85B lacking its leader sequence directed to the apoplast, which reached a yield of 100 mg of antigen per kg of fresh leaf. Glycosylated and non-glycosylated fusion proteins were obtained in the apoplastic and cytoplasmic space, respectively. We showed the feasibility of producing Ag85B-CP protein in N. benthamiana leaves for application as a subunit vaccine and demonstrated the importance of expressing mature Ag85B to increase yield and to avoid the production of degraded protein fragments unsuitable for a pharmaceutical product.
Fil: González, Pablo A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina
Fil: Puccio, Franco Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina
Fil: Zelada, Alicia Mercedes. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina
Materia
Ag85B
Tuberculosis
Molecular pharming
VLPs
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/210774
Acceder
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network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plantsGonzález, Pablo A.Puccio, Franco DamiánZelada, Alicia MercedesAg85BTuberculosisMolecular pharmingVLPshttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3The development of alternative subunit based-vaccines against tuberculosis is necessary due to variable efficiency and some security concerns of the BCG vaccine. The aim of this work was evaluate the production of the Mycobacterium tuberculosis Ag85B antigen fused to Potato Virus X Coat Protein (PVX-CP) by transient expression in Nicotiana benthamiana for subunit-based tuberculosis vaccine formulation. A codon-optimized M. tuberculosis Ag85B gene was fused to PVX-CP and expressed both as a full length precursor and as a mature version lacking the leader peptide. Signal peptides of N. tabacum genes were added to precursor and mature Ag85B-CP to compare the efficiency of cytoplasmic and apoplastic expression. Constructs were agroinfiltrated into N. benthamiana leaves and the yield and integrity of recombinant proteins were analysed. Glycosylation status was determined by treatment with peptide N-glycosidase F. The highest amounts of fusion protein were obtained by expressing mature Ag85B lacking its leader sequence directed to the apoplast, which reached a yield of 100 mg of antigen per kg of fresh leaf. Glycosylated and non-glycosylated fusion proteins were obtained in the apoplastic and cytoplasmic space, respectively. We showed the feasibility of producing Ag85B-CP protein in N. benthamiana leaves for application as a subunit vaccine and demonstrated the importance of expressing mature Ag85B to increase yield and to avoid the production of degraded protein fragments unsuitable for a pharmaceutical product.Fil: González, Pablo A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Puccio, Franco Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Zelada, Alicia Mercedes. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaScience Publications2018-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/210774González, Pablo A.; Puccio, Franco Damián; Zelada, Alicia Mercedes; Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants; Science Publications; American Journal of Biochemistry and Biotechnology; 14; 4; 10-2018; 238-2461553-34681558-6332CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://thescipub.com/abstract/10.3844/ajbbsp.2018.238.246info:eu-repo/semantics/altIdentifier/doi/10.3844/ajbbsp.2018.238.246info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:04:55Zoai:ri.conicet.gov.ar:11336/210774instacron: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:34982025-10-15 15:04:55.377CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
title Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
spellingShingle Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
González, Pablo A.
Ag85B
Tuberculosis
Molecular pharming
VLPs
title_short Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
title_full Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
title_fullStr Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
title_full_unstemmed Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
title_sort Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants
dc.creator.none.fl_str_mv González, Pablo A.
Puccio, Franco Damián
Zelada, Alicia Mercedes
author González, Pablo A.
author_facet González, Pablo A.
Puccio, Franco Damián
Zelada, Alicia Mercedes
author_role author
author2 Puccio, Franco Damián
Zelada, Alicia Mercedes
author2_role author
author
dc.subject.none.fl_str_mv Ag85B
Tuberculosis
Molecular pharming
VLPs
topic Ag85B
Tuberculosis
Molecular pharming
VLPs
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv The development of alternative subunit based-vaccines against tuberculosis is necessary due to variable efficiency and some security concerns of the BCG vaccine. The aim of this work was evaluate the production of the Mycobacterium tuberculosis Ag85B antigen fused to Potato Virus X Coat Protein (PVX-CP) by transient expression in Nicotiana benthamiana for subunit-based tuberculosis vaccine formulation. A codon-optimized M. tuberculosis Ag85B gene was fused to PVX-CP and expressed both as a full length precursor and as a mature version lacking the leader peptide. Signal peptides of N. tabacum genes were added to precursor and mature Ag85B-CP to compare the efficiency of cytoplasmic and apoplastic expression. Constructs were agroinfiltrated into N. benthamiana leaves and the yield and integrity of recombinant proteins were analysed. Glycosylation status was determined by treatment with peptide N-glycosidase F. The highest amounts of fusion protein were obtained by expressing mature Ag85B lacking its leader sequence directed to the apoplast, which reached a yield of 100 mg of antigen per kg of fresh leaf. Glycosylated and non-glycosylated fusion proteins were obtained in the apoplastic and cytoplasmic space, respectively. We showed the feasibility of producing Ag85B-CP protein in N. benthamiana leaves for application as a subunit vaccine and demonstrated the importance of expressing mature Ag85B to increase yield and to avoid the production of degraded protein fragments unsuitable for a pharmaceutical product.
Fil: González, Pablo A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina
Fil: Puccio, Franco Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina
Fil: Zelada, Alicia Mercedes. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina
description The development of alternative subunit based-vaccines against tuberculosis is necessary due to variable efficiency and some security concerns of the BCG vaccine. The aim of this work was evaluate the production of the Mycobacterium tuberculosis Ag85B antigen fused to Potato Virus X Coat Protein (PVX-CP) by transient expression in Nicotiana benthamiana for subunit-based tuberculosis vaccine formulation. A codon-optimized M. tuberculosis Ag85B gene was fused to PVX-CP and expressed both as a full length precursor and as a mature version lacking the leader peptide. Signal peptides of N. tabacum genes were added to precursor and mature Ag85B-CP to compare the efficiency of cytoplasmic and apoplastic expression. Constructs were agroinfiltrated into N. benthamiana leaves and the yield and integrity of recombinant proteins were analysed. Glycosylation status was determined by treatment with peptide N-glycosidase F. The highest amounts of fusion protein were obtained by expressing mature Ag85B lacking its leader sequence directed to the apoplast, which reached a yield of 100 mg of antigen per kg of fresh leaf. Glycosylated and non-glycosylated fusion proteins were obtained in the apoplastic and cytoplasmic space, respectively. We showed the feasibility of producing Ag85B-CP protein in N. benthamiana leaves for application as a subunit vaccine and demonstrated the importance of expressing mature Ag85B to increase yield and to avoid the production of degraded protein fragments unsuitable for a pharmaceutical product.
publishDate 2018
dc.date.none.fl_str_mv 2018-10
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/11336/210774
González, Pablo A.; Puccio, Franco Damián; Zelada, Alicia Mercedes; Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants; Science Publications; American Journal of Biochemistry and Biotechnology; 14; 4; 10-2018; 238-246
1553-3468
1558-6332
CONICET Digital
CONICET
url http://hdl.handle.net/11336/210774
identifier_str_mv González, Pablo A.; Puccio, Franco Damián; Zelada, Alicia Mercedes; Efficient production of glycosylated and non-glycosylated mycobacterium tuberculosis antigen 85B fused to PVX coat protein in Nicotiana benthamiana plants; Science Publications; American Journal of Biochemistry and Biotechnology; 14; 4; 10-2018; 238-246
1553-3468
1558-6332
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://thescipub.com/abstract/10.3844/ajbbsp.2018.238.246
info:eu-repo/semantics/altIdentifier/doi/10.3844/ajbbsp.2018.238.246
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Science Publications
publisher.none.fl_str_mv Science Publications
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 13.22299

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