Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding

Autores
Pavan, María Elisa; Pavan, Esteban E.; Cairo, Fabian Martin; Pettinari, María Julia
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, whichfacilitated its efficient purification by simple washing steps; however, it could not be recognizedby specific antibodies. Refolding conditions were subsequently analyzed in a high-throughputmanner that enabled nearly a hundred different conditions to be tested simultaneously. Therecovery of the ability of PA to be recognized by antibodies was screened by dot blot usinga coefficient that provided a measure of properly refolded protein levels with a high degreeof discrimination. The best refolding conditions resulted in a tenfold increase in the intensityof the dot blot compared to the control. The only refolding additive that consistently yieldedgood results was L-arginine. The statistical analysis identified both cooperative and negativeinteractions between the different refolding additives. The high-throughput approach describedin this study that enabled overproduction, purification and refolding of PA in a simple andstraightforward manner, can be potentially useful for the rapid screening of adequate refoldingconditions for other overexpressed antigenic proteins.
Fil: Pavan, María Elisa. Biochemiq; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina
Fil: Pavan, Esteban E.. Politecnico di Milano; Italia
Fil: Cairo, Fabian Martin. Biochemiq; Argentina
Fil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Materia
Bacillus anthracis
Protective antigen
Protein refolding
High-throughput refolding screening
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:11336/48529
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/48529
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refoldingPavan, María ElisaPavan, Esteban E.Cairo, Fabian MartinPettinari, María JuliaBacillus anthracisProtective antigenProtein refoldingHigh-throughput refolding screeninghttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, whichfacilitated its efficient purification by simple washing steps; however, it could not be recognizedby specific antibodies. Refolding conditions were subsequently analyzed in a high-throughputmanner that enabled nearly a hundred different conditions to be tested simultaneously. Therecovery of the ability of PA to be recognized by antibodies was screened by dot blot usinga coefficient that provided a measure of properly refolded protein levels with a high degreeof discrimination. The best refolding conditions resulted in a tenfold increase in the intensityof the dot blot compared to the control. The only refolding additive that consistently yieldedgood results was L-arginine. The statistical analysis identified both cooperative and negativeinteractions between the different refolding additives. The high-throughput approach describedin this study that enabled overproduction, purification and refolding of PA in a simple andstraightforward manner, can be potentially useful for the rapid screening of adequate refoldingconditions for other overexpressed antigenic proteins.Fil: Pavan, María Elisa. Biochemiq; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Pavan, Esteban E.. Politecnico di Milano; ItaliaFil: Cairo, Fabian Martin. Biochemiq; ArgentinaFil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaAsociación Argentina de Microbiología2016-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/48529Pavan, María Elisa; Pavan, Esteban E.; Cairo, Fabian Martin; Pettinari, María Julia; Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding; Asociación Argentina de Microbiología; Revista Argentina de Microbiología; 48; 1; 1-2016; 5-140325-75411851-7617CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.ram.2015.10.004info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0325754115001509info:eu-repo/semantics/altIdentifier/url/http://www.redalyc.org/articulo.oa?id=213045299002info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:51:30Zoai:ri.conicet.gov.ar:11336/48529instacron: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 09:51:30.355CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
title Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
spellingShingle Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
Pavan, María Elisa
Bacillus anthracis
Protective antigen
Protein refolding
High-throughput refolding screening
title_short Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
title_full Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
title_fullStr Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
title_full_unstemmed Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
title_sort Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
dc.creator.none.fl_str_mv Pavan, María Elisa
Pavan, Esteban E.
Cairo, Fabian Martin
Pettinari, María Julia
author Pavan, María Elisa
author_facet Pavan, María Elisa
Pavan, Esteban E.
Cairo, Fabian Martin
Pettinari, María Julia
author_role author
author2 Pavan, Esteban E.
Cairo, Fabian Martin
Pettinari, María Julia
author2_role author
author
author
dc.subject.none.fl_str_mv Bacillus anthracis
Protective antigen
Protein refolding
High-throughput refolding screening
topic Bacillus anthracis
Protective antigen
Protein refolding
High-throughput refolding screening
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, whichfacilitated its efficient purification by simple washing steps; however, it could not be recognizedby specific antibodies. Refolding conditions were subsequently analyzed in a high-throughputmanner that enabled nearly a hundred different conditions to be tested simultaneously. Therecovery of the ability of PA to be recognized by antibodies was screened by dot blot usinga coefficient that provided a measure of properly refolded protein levels with a high degreeof discrimination. The best refolding conditions resulted in a tenfold increase in the intensityof the dot blot compared to the control. The only refolding additive that consistently yieldedgood results was L-arginine. The statistical analysis identified both cooperative and negativeinteractions between the different refolding additives. The high-throughput approach describedin this study that enabled overproduction, purification and refolding of PA in a simple andstraightforward manner, can be potentially useful for the rapid screening of adequate refoldingconditions for other overexpressed antigenic proteins.
Fil: Pavan, María Elisa. Biochemiq; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina
Fil: Pavan, Esteban E.. Politecnico di Milano; Italia
Fil: Cairo, Fabian Martin. Biochemiq; Argentina
Fil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
description Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, whichfacilitated its efficient purification by simple washing steps; however, it could not be recognizedby specific antibodies. Refolding conditions were subsequently analyzed in a high-throughputmanner that enabled nearly a hundred different conditions to be tested simultaneously. Therecovery of the ability of PA to be recognized by antibodies was screened by dot blot usinga coefficient that provided a measure of properly refolded protein levels with a high degreeof discrimination. The best refolding conditions resulted in a tenfold increase in the intensityof the dot blot compared to the control. The only refolding additive that consistently yieldedgood results was L-arginine. The statistical analysis identified both cooperative and negativeinteractions between the different refolding additives. The high-throughput approach describedin this study that enabled overproduction, purification and refolding of PA in a simple andstraightforward manner, can be potentially useful for the rapid screening of adequate refoldingconditions for other overexpressed antigenic proteins.
publishDate 2016
dc.date.none.fl_str_mv 2016-01
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/11336/48529
Pavan, María Elisa; Pavan, Esteban E.; Cairo, Fabian Martin; Pettinari, María Julia; Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding; Asociación Argentina de Microbiología; Revista Argentina de Microbiología; 48; 1; 1-2016; 5-14
0325-7541
1851-7617
CONICET Digital
CONICET
url http://hdl.handle.net/11336/48529
identifier_str_mv Pavan, María Elisa; Pavan, Esteban E.; Cairo, Fabian Martin; Pettinari, María Julia; Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding; Asociación Argentina de Microbiología; Revista Argentina de Microbiología; 48; 1; 1-2016; 5-14
0325-7541
1851-7617
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ram.2015.10.004
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0325754115001509
info:eu-repo/semantics/altIdentifier/url/http://www.redalyc.org/articulo.oa?id=213045299002
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
dc.publisher.none.fl_str_mv Asociación Argentina de Microbiología
publisher.none.fl_str_mv Asociación Argentina de Microbiología
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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