Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter

Autores
Noseda, Diego Gabriel; Recúpero, Matías Nicolás; Blasco, Martín; Ortiz, Gastón Ezequiel; Galvagno, Miguel Angel
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25 °C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs280 nm and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.
Fil: Noseda, Diego Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Recúpero, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Blasco, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Ortiz, Gastón Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Galvagno, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentina
Materia
BOVINE CHYMOSIN
FED-BATCH FERMENTATION PROCESS
METHANOL-INDUCIBLE AOX1 PROMOTER
OPTIMIZATION
PICHIA (KOMAGATAELLA) PASTORIS
PURIFICATION
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:11336/94900
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/94900
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoterNoseda, Diego GabrielRecúpero, Matías NicolásBlasco, MartínOrtiz, Gastón EzequielGalvagno, Miguel AngelBOVINE CHYMOSINFED-BATCH FERMENTATION PROCESSMETHANOL-INDUCIBLE AOX1 PROMOTEROPTIMIZATIONPICHIA (KOMAGATAELLA) PASTORISPURIFICATIONhttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2https://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25 °C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs280 nm and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.Fil: Noseda, Diego Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Recúpero, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Blasco, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ortiz, Gastón Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Galvagno, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Química; ArgentinaAcademic Press Inc Elsevier Science2013-12info: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/11336/94900Noseda, Diego Gabriel; Recúpero, Matías Nicolás; Blasco, Martín; Ortiz, Gastón Ezequiel; Galvagno, Miguel Angel; Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter; Academic Press Inc Elsevier Science; Protein Expression and Purification; 92; 2; 12-2013; 235-2441046-5928CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S104659281300168Xinfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2013.08.018info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:33:54Zoai:ri.conicet.gov.ar:11336/94900instacron: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 10:33:54.673CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
title Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
spellingShingle Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
Noseda, Diego Gabriel
BOVINE CHYMOSIN
FED-BATCH FERMENTATION PROCESS
METHANOL-INDUCIBLE AOX1 PROMOTER
OPTIMIZATION
PICHIA (KOMAGATAELLA) PASTORIS
PURIFICATION
title_short Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
title_full Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
title_fullStr Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
title_full_unstemmed Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
title_sort Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
dc.creator.none.fl_str_mv Noseda, Diego Gabriel
Recúpero, Matías Nicolás
Blasco, Martín
Ortiz, Gastón Ezequiel
Galvagno, Miguel Angel
author Noseda, Diego Gabriel
author_facet Noseda, Diego Gabriel
Recúpero, Matías Nicolás
Blasco, Martín
Ortiz, Gastón Ezequiel
Galvagno, Miguel Angel
author_role author
author2 Recúpero, Matías Nicolás
Blasco, Martín
Ortiz, Gastón Ezequiel
Galvagno, Miguel Angel
author2_role author
author
author
author
dc.subject.none.fl_str_mv BOVINE CHYMOSIN
FED-BATCH FERMENTATION PROCESS
METHANOL-INDUCIBLE AOX1 PROMOTER
OPTIMIZATION
PICHIA (KOMAGATAELLA) PASTORIS
PURIFICATION
topic BOVINE CHYMOSIN
FED-BATCH FERMENTATION PROCESS
METHANOL-INDUCIBLE AOX1 PROMOTER
OPTIMIZATION
PICHIA (KOMAGATAELLA) PASTORIS
PURIFICATION
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25 °C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs280 nm and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.
Fil: Noseda, Diego Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Recúpero, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Blasco, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Ortiz, Gastón Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Galvagno, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Química; Argentina
description The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25 °C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs280 nm and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.
publishDate 2013
dc.date.none.fl_str_mv 2013-12
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/11336/94900
Noseda, Diego Gabriel; Recúpero, Matías Nicolás; Blasco, Martín; Ortiz, Gastón Ezequiel; Galvagno, Miguel Angel; Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter; Academic Press Inc Elsevier Science; Protein Expression and Purification; 92; 2; 12-2013; 235-244
1046-5928
CONICET Digital
CONICET
url http://hdl.handle.net/11336/94900
identifier_str_mv Noseda, Diego Gabriel; Recúpero, Matías Nicolás; Blasco, Martín; Ortiz, Gastón Ezequiel; Galvagno, Miguel Angel; Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter; Academic Press Inc Elsevier Science; Protein Expression and Purification; 92; 2; 12-2013; 235-244
1046-5928
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://www.sciencedirect.com/science/article/pii/S104659281300168X
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2013.08.018
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 Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
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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