Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies

Autores
Almasia, Natalia Ines; Molinari, Maria Paula; Maroniche, Guillermo Andrés; Nahirñak, Vanesa; Barrios Barón, María Pilar; Taboga, Oscar Alberto; Vazquez Rovere, Cecilia
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanumtuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal andantibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state usingbacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility,misfolding or low yields.Results: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insectcells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinantpeptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. Anoptimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gaverise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stabletransgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinantantimicrobial peptides expression in other heterologous systems.Conclusions: The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide inbaculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploitedfor its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of thebaculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functionalanalysis and antibody production, as an important step to both elucidate their accurate physiological role and todeepen the study of their biotechnological uses.
Fil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Molinari, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Maroniche, Guillermo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Nahirñak, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Barrios Barón, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Taboga, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Vazquez Rovere, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Materia
ANTIMICROBIAL PEPTIDE
CYSTEIN RICH
SNAKIN 1
SPODOPTERA FRUGIPERDA SF9 CELLS
BACULOVIRUS EXPRESSION SYSTEM
GASA
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:11336/43093
Acceder
id CONICETDig_392ec4a340ba17a862dc765973d189fe
oai_identifier_str oai:ri.conicet.gov.ar:11336/43093
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodiesAlmasia, Natalia InesMolinari, Maria PaulaMaroniche, Guillermo AndrésNahirñak, VanesaBarrios Barón, María PilarTaboga, Oscar AlbertoVazquez Rovere, CeciliaANTIMICROBIAL PEPTIDECYSTEIN RICHSNAKIN 1SPODOPTERA FRUGIPERDA SF9 CELLSBACULOVIRUS EXPRESSION SYSTEMGASAhttps://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4Background: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanumtuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal andantibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state usingbacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility,misfolding or low yields.Results: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insectcells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinantpeptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. Anoptimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gaverise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stabletransgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinantantimicrobial peptides expression in other heterologous systems.Conclusions: The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide inbaculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploitedfor its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of thebaculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functionalanalysis and antibody production, as an important step to both elucidate their accurate physiological role and todeepen the study of their biotechnological uses.Fil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Molinari, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Maroniche, Guillermo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Nahirñak, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Barrios Barón, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Taboga, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Vazquez Rovere, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaBioMed Central2017-11info: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/11336/43093Almasia, Natalia Ines; Molinari, Maria Paula; Maroniche, Guillermo Andrés; Nahirñak, Vanesa; Barrios Barón, María Pilar; et al.; Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies; BioMed Central; Bmc Biotechnology; 17; 1; 11-20171472-6750CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-017-0401-2info:eu-repo/semantics/altIdentifier/doi/10.1186/s12896-017-0401-2info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:02:59Zoai:ri.conicet.gov.ar:11336/43093instacron: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 10:02:59.887CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
title Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
spellingShingle Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
Almasia, Natalia Ines
ANTIMICROBIAL PEPTIDE
CYSTEIN RICH
SNAKIN 1
SPODOPTERA FRUGIPERDA SF9 CELLS
BACULOVIRUS EXPRESSION SYSTEM
GASA
title_short Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
title_full Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
title_fullStr Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
title_full_unstemmed Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
title_sort Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies
dc.creator.none.fl_str_mv Almasia, Natalia Ines
Molinari, Maria Paula
Maroniche, Guillermo Andrés
Nahirñak, Vanesa
Barrios Barón, María Pilar
Taboga, Oscar Alberto
Vazquez Rovere, Cecilia
author Almasia, Natalia Ines
author_facet Almasia, Natalia Ines
Molinari, Maria Paula
Maroniche, Guillermo Andrés
Nahirñak, Vanesa
Barrios Barón, María Pilar
Taboga, Oscar Alberto
Vazquez Rovere, Cecilia
author_role author
author2 Molinari, Maria Paula
Maroniche, Guillermo Andrés
Nahirñak, Vanesa
Barrios Barón, María Pilar
Taboga, Oscar Alberto
Vazquez Rovere, Cecilia
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv ANTIMICROBIAL PEPTIDE
CYSTEIN RICH
SNAKIN 1
SPODOPTERA FRUGIPERDA SF9 CELLS
BACULOVIRUS EXPRESSION SYSTEM
GASA
topic ANTIMICROBIAL PEPTIDE
CYSTEIN RICH
SNAKIN 1
SPODOPTERA FRUGIPERDA SF9 CELLS
BACULOVIRUS EXPRESSION SYSTEM
GASA
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv Background: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanumtuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal andantibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state usingbacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility,misfolding or low yields.Results: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insectcells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinantpeptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. Anoptimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gaverise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stabletransgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinantantimicrobial peptides expression in other heterologous systems.Conclusions: The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide inbaculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploitedfor its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of thebaculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functionalanalysis and antibody production, as an important step to both elucidate their accurate physiological role and todeepen the study of their biotechnological uses.
Fil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Molinari, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Maroniche, Guillermo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Nahirñak, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Barrios Barón, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Taboga, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
Fil: Vazquez Rovere, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina
description Background: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanumtuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal andantibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state usingbacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility,misfolding or low yields.Results: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insectcells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinantpeptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. Anoptimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gaverise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stabletransgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinantantimicrobial peptides expression in other heterologous systems.Conclusions: The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide inbaculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploitedfor its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of thebaculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functionalanalysis and antibody production, as an important step to both elucidate their accurate physiological role and todeepen the study of their biotechnological uses.
publishDate 2017
dc.date.none.fl_str_mv 2017-11
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/11336/43093
Almasia, Natalia Ines; Molinari, Maria Paula; Maroniche, Guillermo Andrés; Nahirñak, Vanesa; Barrios Barón, María Pilar; et al.; Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies; BioMed Central; Bmc Biotechnology; 17; 1; 11-2017
1472-6750
CONICET Digital
CONICET
url http://hdl.handle.net/11336/43093
identifier_str_mv Almasia, Natalia Ines; Molinari, Maria Paula; Maroniche, Guillermo Andrés; Nahirñak, Vanesa; Barrios Barón, María Pilar; et al.; Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies; BioMed Central; Bmc Biotechnology; 17; 1; 11-2017
1472-6750
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://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-017-0401-2
info:eu-repo/semantics/altIdentifier/doi/10.1186/s12896-017-0401-2
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 BioMed Central
publisher.none.fl_str_mv BioMed Central
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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