Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12

Autores
Morales Álvarez, Edwin David; Rivera Hoyos, Claudia Marcela; Baena Moncada, Angelica Maria; Landázuri, Patricia; Poutou Piñales, Raúl A.; Sáenz Suárez, Homero; Barrera, Luis A.; Echeverri Peña, Olga Y.
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78. 13 to 94. 35 ng/ml and the specific activity varied from 34. 20 to 25. 97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli. © 2013 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
Fil: Morales Álvarez, Edwin David. Universidad del Quindio; Colombia
Fil: Rivera Hoyos, Claudia Marcela. Universidad del Quindio. Facultad de Medicina; Colombia
Fil: Baena Moncada, Angelica Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Landázuri, Patricia. Universidad del Quindio. Facultad de Medicina. Centro de Investig. Biomédicas; Colombia
Fil: Poutou Piñales, Raúl A.. Pontificia Universidad Javeriana; Colombia
Fil: Sáenz Suárez, Homero. Universidad del Quindio; Colombia
Fil: Barrera, Luis A.. Pontificia Universidad Javeriana; Colombia
Fil: Echeverri Peña, Olga Y.. Pontificia Universidad Javeriana; Colombia
Materia
AFFINITY CHROMATOGRAPHY
E. COLI DH5Α
IDURONATE 2-SULFATE SULFATASE
RECOMBINANT PROTEIN EXPRESSION
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:11336/213588
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/213588
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12Morales Álvarez, Edwin DavidRivera Hoyos, Claudia MarcelaBaena Moncada, Angelica MariaLandázuri, PatriciaPoutou Piñales, Raúl A.Sáenz Suárez, HomeroBarrera, Luis A.Echeverri Peña, Olga Y.AFFINITY CHROMATOGRAPHYE. COLI DH5ΑIDURONATE 2-SULFATE SULFATASERECOMBINANT PROTEIN EXPRESSIONhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78. 13 to 94. 35 ng/ml and the specific activity varied from 34. 20 to 25. 97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli. © 2013 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.Fil: Morales Álvarez, Edwin David. Universidad del Quindio; ColombiaFil: Rivera Hoyos, Claudia Marcela. Universidad del Quindio. Facultad de Medicina; ColombiaFil: Baena Moncada, Angelica Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Landázuri, Patricia. Universidad del Quindio. Facultad de Medicina. Centro de Investig. Biomédicas; ColombiaFil: Poutou Piñales, Raúl A.. Pontificia Universidad Javeriana; ColombiaFil: Sáenz Suárez, Homero. Universidad del Quindio; ColombiaFil: Barrera, Luis A.. Pontificia Universidad Javeriana; ColombiaFil: Echeverri Peña, Olga Y.. Pontificia Universidad Javeriana; ColombiaKorean Society Microbiology2013-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/213588Morales Álvarez, Edwin David; Rivera Hoyos, Claudia Marcela; Baena Moncada, Angelica Maria; Landázuri, Patricia; Poutou Piñales, Raúl A.; et al.; Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12; Korean Society Microbiology; Journal of Microbiology and Biotechnology; 51; 2; 4-2013; 213-2211225-8873CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1007/s12275-013-2416-2info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:26:21Zoai:ri.conicet.gov.ar:11336/213588instacron: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 11:26:21.986CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
title Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
spellingShingle Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
Morales Álvarez, Edwin David
AFFINITY CHROMATOGRAPHY
E. COLI DH5Α
IDURONATE 2-SULFATE SULFATASE
RECOMBINANT PROTEIN EXPRESSION
title_short Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
title_full Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
title_fullStr Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
title_full_unstemmed Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
title_sort Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
dc.creator.none.fl_str_mv Morales Álvarez, Edwin David
Rivera Hoyos, Claudia Marcela
Baena Moncada, Angelica Maria
Landázuri, Patricia
Poutou Piñales, Raúl A.
Sáenz Suárez, Homero
Barrera, Luis A.
Echeverri Peña, Olga Y.
author Morales Álvarez, Edwin David
author_facet Morales Álvarez, Edwin David
Rivera Hoyos, Claudia Marcela
Baena Moncada, Angelica Maria
Landázuri, Patricia
Poutou Piñales, Raúl A.
Sáenz Suárez, Homero
Barrera, Luis A.
Echeverri Peña, Olga Y.
author_role author
author2 Rivera Hoyos, Claudia Marcela
Baena Moncada, Angelica Maria
Landázuri, Patricia
Poutou Piñales, Raúl A.
Sáenz Suárez, Homero
Barrera, Luis A.
Echeverri Peña, Olga Y.
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv AFFINITY CHROMATOGRAPHY
E. COLI DH5Α
IDURONATE 2-SULFATE SULFATASE
RECOMBINANT PROTEIN EXPRESSION
topic AFFINITY CHROMATOGRAPHY
E. COLI DH5Α
IDURONATE 2-SULFATE SULFATASE
RECOMBINANT PROTEIN EXPRESSION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78. 13 to 94. 35 ng/ml and the specific activity varied from 34. 20 to 25. 97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli. © 2013 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
Fil: Morales Álvarez, Edwin David. Universidad del Quindio; Colombia
Fil: Rivera Hoyos, Claudia Marcela. Universidad del Quindio. Facultad de Medicina; Colombia
Fil: Baena Moncada, Angelica Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Landázuri, Patricia. Universidad del Quindio. Facultad de Medicina. Centro de Investig. Biomédicas; Colombia
Fil: Poutou Piñales, Raúl A.. Pontificia Universidad Javeriana; Colombia
Fil: Sáenz Suárez, Homero. Universidad del Quindio; Colombia
Fil: Barrera, Luis A.. Pontificia Universidad Javeriana; Colombia
Fil: Echeverri Peña, Olga Y.. Pontificia Universidad Javeriana; Colombia
description The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78. 13 to 94. 35 ng/ml and the specific activity varied from 34. 20 to 25. 97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli. © 2013 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
publishDate 2013
dc.date.none.fl_str_mv 2013-04
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/11336/213588
Morales Álvarez, Edwin David; Rivera Hoyos, Claudia Marcela; Baena Moncada, Angelica Maria; Landázuri, Patricia; Poutou Piñales, Raúl A.; et al.; Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12; Korean Society Microbiology; Journal of Microbiology and Biotechnology; 51; 2; 4-2013; 213-221
1225-8873
CONICET Digital
CONICET
url http://hdl.handle.net/11336/213588
identifier_str_mv Morales Álvarez, Edwin David; Rivera Hoyos, Claudia Marcela; Baena Moncada, Angelica Maria; Landázuri, Patricia; Poutou Piñales, Raúl A.; et al.; Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12; Korean Society Microbiology; Journal of Microbiology and Biotechnology; 51; 2; 4-2013; 213-221
1225-8873
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1007/s12275-013-2416-2
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 Korean Society Microbiology
publisher.none.fl_str_mv Korean Society 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
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score 12.982451

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