Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin

Autores
Curto, Pedro; Lufrano, Daniela; Pinto, Cátia; Custódio, Valéria; Gomes, Ana Catarina; Trejo, Sebastian Alejandro; Bakas, Laura Susana; Vairo Cavalli, Sandra Elizabeth; Faro, Carlos; Simões, Isaura
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Typical plant aspartic protease zymogens comprise a characteristic and plant specific insert (PSI). PSI domains can interact with membranes and a role as defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet, due to existing problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the Generally Regarded as Safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI as well as its non-glycosylated mutant. A purification protocol was established with protein yields of ∼4.0 mg/L for both wild-type and non-glycosylated PSI which represents the highest reported yield for a non-tagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its anti-fungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.
Fil: Curto, Pedro. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; Portugal
Fil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Pinto, Cátia. Biocant; Portugal
Fil: Custódio, Valéria. Biocant; Portugal
Fil: Gomes, Ana Catarina. Biocant; Portugal
Fil: Trejo, Sebastian Alejandro. Universitat Autònoma de Barcelona; España
Fil: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Vairo Cavalli, Sandra Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Faro, Carlos. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; Portugal
Fil: Simões, Isaura. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; Portugal
Materia
Cirsin
Plant Aspartic Proteases
Plant Specific Insert (Psi)
Kluyveromyces Lactis
Heterologous Expression
Antimicrobial Activity
Antimicrobial Peptides
Saposin-Like Domain
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:11336/80536
Acceder
id CONICETDig_0b52f5dbaad81ad4fbf6612e00b3ce46
oai_identifier_str oai:ri.conicet.gov.ar:11336/80536
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsinCurto, PedroLufrano, DanielaPinto, CátiaCustódio, ValériaGomes, Ana CatarinaTrejo, Sebastian AlejandroBakas, Laura SusanaVairo Cavalli, Sandra ElizabethFaro, CarlosSimões, IsauraCirsinPlant Aspartic ProteasesPlant Specific Insert (Psi)Kluyveromyces LactisHeterologous ExpressionAntimicrobial ActivityAntimicrobial PeptidesSaposin-Like Domainhttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2Typical plant aspartic protease zymogens comprise a characteristic and plant specific insert (PSI). PSI domains can interact with membranes and a role as defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet, due to existing problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the Generally Regarded as Safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI as well as its non-glycosylated mutant. A purification protocol was established with protein yields of ∼4.0 mg/L for both wild-type and non-glycosylated PSI which represents the highest reported yield for a non-tagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its anti-fungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.Fil: Curto, Pedro. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; PortugalFil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaFil: Pinto, Cátia. Biocant; PortugalFil: Custódio, Valéria. Biocant; PortugalFil: Gomes, Ana Catarina. Biocant; PortugalFil: Trejo, Sebastian Alejandro. Universitat Autònoma de Barcelona; EspañaFil: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaFil: Vairo Cavalli, Sandra Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaFil: Faro, Carlos. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; PortugalFil: Simões, Isaura. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; PortugalAmerican Society for Microbiology2014-01info: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/11336/80536Curto, Pedro; Lufrano, Daniela; Pinto, Cátia; Custódio, Valéria; Gomes, Ana Catarina; et al.; Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin; American Society for Microbiology; Applied And Environmental Microbiology; 80; 1; 1-2014; 86-960099-2240CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://aem.asm.org/content/early/2013/10/07/AEM.03151-13.longinfo:eu-repo/semantics/altIdentifier/doi/10.1128/AEM.03151-13info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:29:33Zoai:ri.conicet.gov.ar:11336/80536instacron: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:34982025-10-22 11:29:33.457CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
title Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
spellingShingle Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
Curto, Pedro
Cirsin
Plant Aspartic Proteases
Plant Specific Insert (Psi)
Kluyveromyces Lactis
Heterologous Expression
Antimicrobial Activity
Antimicrobial Peptides
Saposin-Like Domain
title_short Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
title_full Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
title_fullStr Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
title_full_unstemmed Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
title_sort Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin
dc.creator.none.fl_str_mv Curto, Pedro
Lufrano, Daniela
Pinto, Cátia
Custódio, Valéria
Gomes, Ana Catarina
Trejo, Sebastian Alejandro
Bakas, Laura Susana
Vairo Cavalli, Sandra Elizabeth
Faro, Carlos
Simões, Isaura
author Curto, Pedro
author_facet Curto, Pedro
Lufrano, Daniela
Pinto, Cátia
Custódio, Valéria
Gomes, Ana Catarina
Trejo, Sebastian Alejandro
Bakas, Laura Susana
Vairo Cavalli, Sandra Elizabeth
Faro, Carlos
Simões, Isaura
author_role author
author2 Lufrano, Daniela
Pinto, Cátia
Custódio, Valéria
Gomes, Ana Catarina
Trejo, Sebastian Alejandro
Bakas, Laura Susana
Vairo Cavalli, Sandra Elizabeth
Faro, Carlos
Simões, Isaura
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Cirsin
Plant Aspartic Proteases
Plant Specific Insert (Psi)
Kluyveromyces Lactis
Heterologous Expression
Antimicrobial Activity
Antimicrobial Peptides
Saposin-Like Domain
topic Cirsin
Plant Aspartic Proteases
Plant Specific Insert (Psi)
Kluyveromyces Lactis
Heterologous Expression
Antimicrobial Activity
Antimicrobial Peptides
Saposin-Like Domain
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv Typical plant aspartic protease zymogens comprise a characteristic and plant specific insert (PSI). PSI domains can interact with membranes and a role as defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet, due to existing problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the Generally Regarded as Safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI as well as its non-glycosylated mutant. A purification protocol was established with protein yields of ∼4.0 mg/L for both wild-type and non-glycosylated PSI which represents the highest reported yield for a non-tagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its anti-fungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.
Fil: Curto, Pedro. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; Portugal
Fil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Pinto, Cátia. Biocant; Portugal
Fil: Custódio, Valéria. Biocant; Portugal
Fil: Gomes, Ana Catarina. Biocant; Portugal
Fil: Trejo, Sebastian Alejandro. Universitat Autònoma de Barcelona; España
Fil: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Vairo Cavalli, Sandra Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Faro, Carlos. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; Portugal
Fil: Simões, Isaura. Universidad de Coimbra. Facultad de Ciencias E Tecnología. Departamento de Ciencias Da Vida; Portugal
description Typical plant aspartic protease zymogens comprise a characteristic and plant specific insert (PSI). PSI domains can interact with membranes and a role as defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet, due to existing problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the Generally Regarded as Safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI as well as its non-glycosylated mutant. A purification protocol was established with protein yields of ∼4.0 mg/L for both wild-type and non-glycosylated PSI which represents the highest reported yield for a non-tagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its anti-fungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.
publishDate 2014
dc.date.none.fl_str_mv 2014-01
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/11336/80536
Curto, Pedro; Lufrano, Daniela; Pinto, Cátia; Custódio, Valéria; Gomes, Ana Catarina; et al.; Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin; American Society for Microbiology; Applied And Environmental Microbiology; 80; 1; 1-2014; 86-96
0099-2240
CONICET Digital
CONICET
url http://hdl.handle.net/11336/80536
identifier_str_mv Curto, Pedro; Lufrano, Daniela; Pinto, Cátia; Custódio, Valéria; Gomes, Ana Catarina; et al.; Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin; American Society for Microbiology; Applied And Environmental Microbiology; 80; 1; 1-2014; 86-96
0099-2240
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://aem.asm.org/content/early/2013/10/07/AEM.03151-13.long
info:eu-repo/semantics/altIdentifier/doi/10.1128/AEM.03151-13
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 American Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
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
_version_ 1846781882026426368
score 12.982451

Publicaciones similares