Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase

Autores
Beassoni, Paola Rita; Pérez de Berti, Federico Javier; Otero, Lisandro Horacio; Risso, Valeria Alejandra; Ferreyra, Raul Gabriel; Lisa, Angela Teresita; Domenech, Carlos Eduardo; Ermácora, Mario R.
Año de publicación
2010
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure. © 2010.
Fil: Beassoni, Paola Rita. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Pérez de Berti, Federico Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Otero, Lisandro Horacio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Ferreyra, Raul Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Lisa, Angela Teresita. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Domenech, Carlos Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Ermácora, Mario R.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
CIRCULAR DICHROISM
FLUORESCENCE
METAL BINDING
PHOSPHORYLCHOLINE PHOSPHATASE
PROTEIN FOLDING
PSEUDOMONAS AERUGINOSA
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:11336/131909
Acceder
id CONICETDig_386c38f522a129e0fb41fefbb7c5b28e
oai_identifier_str oai:ri.conicet.gov.ar:11336/131909
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphataseBeassoni, Paola RitaPérez de Berti, Federico JavierOtero, Lisandro HoracioRisso, Valeria AlejandraFerreyra, Raul GabrielLisa, Angela TeresitaDomenech, Carlos EduardoErmácora, Mario R.CIRCULAR DICHROISMFLUORESCENCEMETAL BINDINGPHOSPHORYLCHOLINE PHOSPHATASEPROTEIN FOLDINGPSEUDOMONAS AERUGINOSAhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure. © 2010.Fil: Beassoni, Paola Rita. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Pérez de Berti, Federico Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Otero, Lisandro Horacio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferreyra, Raul Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lisa, Angela Teresita. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Domenech, Carlos Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Ermácora, Mario R.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaAcademic Press Inc Elsevier Science2010-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/131909Beassoni, Paola Rita; Pérez de Berti, Federico Javier; Otero, Lisandro Horacio; Risso, Valeria Alejandra; Ferreyra, Raul Gabriel; et al.; Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase; Academic Press Inc Elsevier Science; Protein Expression and Purification; 71; 2; 6-2010; 153-1591046-5928CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2010.01.006info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:11:50Zoai:ri.conicet.gov.ar:11336/131909instacron: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:34982025-10-15 15:11:51.024CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
title Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
spellingShingle Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
Beassoni, Paola Rita
CIRCULAR DICHROISM
FLUORESCENCE
METAL BINDING
PHOSPHORYLCHOLINE PHOSPHATASE
PROTEIN FOLDING
PSEUDOMONAS AERUGINOSA
title_short Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
title_full Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
title_fullStr Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
title_full_unstemmed Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
title_sort Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase
dc.creator.none.fl_str_mv Beassoni, Paola Rita
Pérez de Berti, Federico Javier
Otero, Lisandro Horacio
Risso, Valeria Alejandra
Ferreyra, Raul Gabriel
Lisa, Angela Teresita
Domenech, Carlos Eduardo
Ermácora, Mario R.
author Beassoni, Paola Rita
author_facet Beassoni, Paola Rita
Pérez de Berti, Federico Javier
Otero, Lisandro Horacio
Risso, Valeria Alejandra
Ferreyra, Raul Gabriel
Lisa, Angela Teresita
Domenech, Carlos Eduardo
Ermácora, Mario R.
author_role author
author2 Pérez de Berti, Federico Javier
Otero, Lisandro Horacio
Risso, Valeria Alejandra
Ferreyra, Raul Gabriel
Lisa, Angela Teresita
Domenech, Carlos Eduardo
Ermácora, Mario R.
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv CIRCULAR DICHROISM
FLUORESCENCE
METAL BINDING
PHOSPHORYLCHOLINE PHOSPHATASE
PROTEIN FOLDING
PSEUDOMONAS AERUGINOSA
topic CIRCULAR DICHROISM
FLUORESCENCE
METAL BINDING
PHOSPHORYLCHOLINE PHOSPHATASE
PROTEIN FOLDING
PSEUDOMONAS AERUGINOSA
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure. © 2010.
Fil: Beassoni, Paola Rita. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Pérez de Berti, Federico Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Otero, Lisandro Horacio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Ferreyra, Raul Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Lisa, Angela Teresita. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Domenech, Carlos Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Ermácora, Mario R.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure. © 2010.
publishDate 2010
dc.date.none.fl_str_mv 2010-06
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/11336/131909
Beassoni, Paola Rita; Pérez de Berti, Federico Javier; Otero, Lisandro Horacio; Risso, Valeria Alejandra; Ferreyra, Raul Gabriel; et al.; Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase; Academic Press Inc Elsevier Science; Protein Expression and Purification; 71; 2; 6-2010; 153-159
1046-5928
CONICET Digital
CONICET
url http://hdl.handle.net/11336/131909
identifier_str_mv Beassoni, Paola Rita; Pérez de Berti, Federico Javier; Otero, Lisandro Horacio; Risso, Valeria Alejandra; Ferreyra, Raul Gabriel; et al.; Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase; Academic Press Inc Elsevier Science; Protein Expression and Purification; 71; 2; 6-2010; 153-159
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/doi/
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2010.01.006
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
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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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