Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems

Autores
Targovnik, Alexandra Marisa; Cascone, Osvaldo; Miranda, María Victoria
Año de publicación
2012
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Aqueous two-phase systems (ATPS) have not yet been applied to purify proteins expressed in insect larvae infected by recombinant baculovirus. This work describes the behavior of typical contaminants in the baculovirus-insect larvae expression system such as larval proteins and baculovirus particles in PEG/phosphate ATPSs, in addition to the extraction and purification of the target protein (horseradish peroxidase isozyme C, HRPC). After assessing the influence of PEG molecular weight, system pH and added salt on the partition constants of HRPC and total protein of a clarified larvae extract, two ATPSs were selected for the first extraction step: PEG 1500/phosphate, pH 7.0 with 4.0 % NaCl (System 1) and PEG/phosphate, pH 5.0 in the absence of NaCl (System 2). Both systems were found to be appropriate since a clarified enzyme-enriched top phase was obtained with a yield of 99 and 90 % respectively. The direct partition of larvae homogenized with the components of systems 1 and 2, yielded a HRPC recovery in top phase of 71.4% and 81.1% respectively, whereas total protein recovery was 5.2% and 3.3% respectively. In both systems, the top phase was clear and particulate material remained in the interphase and the bottom phase. The bulk of immunogenic proteins of the larvae concentrated in the bottom phase of both systems. The PCR assay revealed the presence of viral DNA in both phases.             It was possible to extract the HRPC back from the PEG-rich phase by adding a fresh magnesium sulphate solution to form a new ATPS, achieving a recovery in the bottom phase of 50% and 98% in Systems 1 and 2 respectively, whereas the recovery of total protein was 69 % and 24 % respectively. The HRPC global recovery of the two-step processes was 35.4 % and 79.6 % for Systems 1 and 2, with purification factors of 14.5 and 114.2 respectively. The final product was free of viral particles.
Fil: Targovnik, Alexandra Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina
Fil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina
Fil: Miranda, María Victoria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina
Materia
HORSERADISH PEROXIDASE
AQUEOUS TWO PHASE SYSTEM
RACHIPLUSIA NU
EXPRESSION AND 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:11336/109254
Acceder
id CONICETDig_993f9f2e65272e3112ec6b6d458e0eef
oai_identifier_str oai:ri.conicet.gov.ar:11336/109254
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systemsTargovnik, Alexandra MarisaCascone, OsvaldoMiranda, María VictoriaHORSERADISH PEROXIDASEAQUEOUS TWO PHASE SYSTEMRACHIPLUSIA NUEXPRESSION AND PURIFICATIONhttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2Aqueous two-phase systems (ATPS) have not yet been applied to purify proteins expressed in insect larvae infected by recombinant baculovirus. This work describes the behavior of typical contaminants in the baculovirus-insect larvae expression system such as larval proteins and baculovirus particles in PEG/phosphate ATPSs, in addition to the extraction and purification of the target protein (horseradish peroxidase isozyme C, HRPC). After assessing the influence of PEG molecular weight, system pH and added salt on the partition constants of HRPC and total protein of a clarified larvae extract, two ATPSs were selected for the first extraction step: PEG 1500/phosphate, pH 7.0 with 4.0 % NaCl (System 1) and PEG/phosphate, pH 5.0 in the absence of NaCl (System 2). Both systems were found to be appropriate since a clarified enzyme-enriched top phase was obtained with a yield of 99 and 90 % respectively. The direct partition of larvae homogenized with the components of systems 1 and 2, yielded a HRPC recovery in top phase of 71.4% and 81.1% respectively, whereas total protein recovery was 5.2% and 3.3% respectively. In both systems, the top phase was clear and particulate material remained in the interphase and the bottom phase. The bulk of immunogenic proteins of the larvae concentrated in the bottom phase of both systems. The PCR assay revealed the presence of viral DNA in both phases.             It was possible to extract the HRPC back from the PEG-rich phase by adding a fresh magnesium sulphate solution to form a new ATPS, achieving a recovery in the bottom phase of 50% and 98% in Systems 1 and 2 respectively, whereas the recovery of total protein was 69 % and 24 % respectively. The HRPC global recovery of the two-step processes was 35.4 % and 79.6 % for Systems 1 and 2, with purification factors of 14.5 and 114.2 respectively. The final product was free of viral particles.Fil: Targovnik, Alexandra Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Miranda, María Victoria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaElsevier Science2012-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/109254Targovnik, Alexandra Marisa; Cascone, Osvaldo; Miranda, María Victoria; Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems; Elsevier Science; Separation and Purification Technology; 98; 9-2012; 199-2051383-5866CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1383586612004340info:eu-repo/semantics/altIdentifier/doi/10.1016/j.seppur.2012.08.004info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:03:06Zoai:ri.conicet.gov.ar:11336/109254instacron: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 10:03:07.16CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
title Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
spellingShingle Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
Targovnik, Alexandra Marisa
HORSERADISH PEROXIDASE
AQUEOUS TWO PHASE SYSTEM
RACHIPLUSIA NU
EXPRESSION AND PURIFICATION
title_short Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
title_full Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
title_fullStr Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
title_full_unstemmed Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
title_sort Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems
dc.creator.none.fl_str_mv Targovnik, Alexandra Marisa
Cascone, Osvaldo
Miranda, María Victoria
author Targovnik, Alexandra Marisa
author_facet Targovnik, Alexandra Marisa
Cascone, Osvaldo
Miranda, María Victoria
author_role author
author2 Cascone, Osvaldo
Miranda, María Victoria
author2_role author
author
dc.subject.none.fl_str_mv HORSERADISH PEROXIDASE
AQUEOUS TWO PHASE SYSTEM
RACHIPLUSIA NU
EXPRESSION AND PURIFICATION
topic HORSERADISH PEROXIDASE
AQUEOUS TWO PHASE SYSTEM
RACHIPLUSIA NU
EXPRESSION AND PURIFICATION
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv Aqueous two-phase systems (ATPS) have not yet been applied to purify proteins expressed in insect larvae infected by recombinant baculovirus. This work describes the behavior of typical contaminants in the baculovirus-insect larvae expression system such as larval proteins and baculovirus particles in PEG/phosphate ATPSs, in addition to the extraction and purification of the target protein (horseradish peroxidase isozyme C, HRPC). After assessing the influence of PEG molecular weight, system pH and added salt on the partition constants of HRPC and total protein of a clarified larvae extract, two ATPSs were selected for the first extraction step: PEG 1500/phosphate, pH 7.0 with 4.0 % NaCl (System 1) and PEG/phosphate, pH 5.0 in the absence of NaCl (System 2). Both systems were found to be appropriate since a clarified enzyme-enriched top phase was obtained with a yield of 99 and 90 % respectively. The direct partition of larvae homogenized with the components of systems 1 and 2, yielded a HRPC recovery in top phase of 71.4% and 81.1% respectively, whereas total protein recovery was 5.2% and 3.3% respectively. In both systems, the top phase was clear and particulate material remained in the interphase and the bottom phase. The bulk of immunogenic proteins of the larvae concentrated in the bottom phase of both systems. The PCR assay revealed the presence of viral DNA in both phases.             It was possible to extract the HRPC back from the PEG-rich phase by adding a fresh magnesium sulphate solution to form a new ATPS, achieving a recovery in the bottom phase of 50% and 98% in Systems 1 and 2 respectively, whereas the recovery of total protein was 69 % and 24 % respectively. The HRPC global recovery of the two-step processes was 35.4 % and 79.6 % for Systems 1 and 2, with purification factors of 14.5 and 114.2 respectively. The final product was free of viral particles.
Fil: Targovnik, Alexandra Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina
Fil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina
Fil: Miranda, María Victoria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina
description Aqueous two-phase systems (ATPS) have not yet been applied to purify proteins expressed in insect larvae infected by recombinant baculovirus. This work describes the behavior of typical contaminants in the baculovirus-insect larvae expression system such as larval proteins and baculovirus particles in PEG/phosphate ATPSs, in addition to the extraction and purification of the target protein (horseradish peroxidase isozyme C, HRPC). After assessing the influence of PEG molecular weight, system pH and added salt on the partition constants of HRPC and total protein of a clarified larvae extract, two ATPSs were selected for the first extraction step: PEG 1500/phosphate, pH 7.0 with 4.0 % NaCl (System 1) and PEG/phosphate, pH 5.0 in the absence of NaCl (System 2). Both systems were found to be appropriate since a clarified enzyme-enriched top phase was obtained with a yield of 99 and 90 % respectively. The direct partition of larvae homogenized with the components of systems 1 and 2, yielded a HRPC recovery in top phase of 71.4% and 81.1% respectively, whereas total protein recovery was 5.2% and 3.3% respectively. In both systems, the top phase was clear and particulate material remained in the interphase and the bottom phase. The bulk of immunogenic proteins of the larvae concentrated in the bottom phase of both systems. The PCR assay revealed the presence of viral DNA in both phases.             It was possible to extract the HRPC back from the PEG-rich phase by adding a fresh magnesium sulphate solution to form a new ATPS, achieving a recovery in the bottom phase of 50% and 98% in Systems 1 and 2 respectively, whereas the recovery of total protein was 69 % and 24 % respectively. The HRPC global recovery of the two-step processes was 35.4 % and 79.6 % for Systems 1 and 2, with purification factors of 14.5 and 114.2 respectively. The final product was free of viral particles.
publishDate 2012
dc.date.none.fl_str_mv 2012-09
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/11336/109254
Targovnik, Alexandra Marisa; Cascone, Osvaldo; Miranda, María Victoria; Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems; Elsevier Science; Separation and Purification Technology; 98; 9-2012; 199-205
1383-5866
CONICET Digital
CONICET
url http://hdl.handle.net/11336/109254
identifier_str_mv Targovnik, Alexandra Marisa; Cascone, Osvaldo; Miranda, María Victoria; Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems; Elsevier Science; Separation and Purification Technology; 98; 9-2012; 199-205
1383-5866
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/S1383586612004340
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.seppur.2012.08.004
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
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv 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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