Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom

Detalhes bibliográficos
Autor(a) principal: Bazon, Murilo Luiz [UNESP]
Data de Publicação: 2017
Outros Autores: Perez-Riverol, Amilcar [UNESP], Dos Santos-Pinto, José Roberto Aparecido [UNESP], Fernandes, Luis Gustavo Romani, Lasa, Alexis Musacchio, Justo-Jacomini, Débora Laís [UNESP], Palma, Mario Sergio [UNESP], De Lima Zollner, Ricardo, Brochetto-Braga, Márcia Regina [UNESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.3390/toxins9090259
http://hdl.handle.net/11449/175114
Resumo: Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.
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spelling Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venomAllergyAntigen 5DiagnosisHeterologous expressionPolybia paulistaPolybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaCentro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaLaboratório de Imunologia Translacional-LIT Departamento de Clínica Médica Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Tessália Vieira de Camargo n◦ 126, Cidade Universitária “Zeferino Vaz”System Biology Department Biomedical Research Division Center for Genetic Engineering and Biotechnology, Ave. 31, e/158 and 190, P.O. Box 6162, CubanacanCentro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental LageadoLaboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaCentro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaCentro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental LageadoFAPESP: 2013/26451-9FAPESP: 2014/13936-7CNPq: 455422/2014-1Universidade Estadual Paulista (Unesp)Universidade Estadual de Campinas (UNICAMP)Center for Genetic Engineering and BiotechnologyBazon, Murilo Luiz [UNESP]Perez-Riverol, Amilcar [UNESP]Dos Santos-Pinto, José Roberto Aparecido [UNESP]Fernandes, Luis Gustavo RomaniLasa, Alexis MusacchioJusto-Jacomini, Débora Laís [UNESP]Palma, Mario Sergio [UNESP]De Lima Zollner, RicardoBrochetto-Braga, Márcia Regina [UNESP]2018-12-11T17:14:26Z2018-12-11T17:14:26Z2017-09-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.3390/toxins9090259Toxins, v. 9, n. 9, 2017.2072-6651http://hdl.handle.net/11449/17511410.3390/toxins90902592-s2.0-850285917462-s2.0-85028591746.pdf2901888624506535Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengToxins0,955info:eu-repo/semantics/openAccess2023-11-22T06:15:10Zoai:repositorio.unesp.br:11449/175114Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T18:25:36.212183Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
title Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
spellingShingle Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
Bazon, Murilo Luiz [UNESP]
Allergy
Antigen 5
Diagnosis
Heterologous expression
Polybia paulista
title_short Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
title_full Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
title_fullStr Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
title_full_unstemmed Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
title_sort Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
author Bazon, Murilo Luiz [UNESP]
author_facet Bazon, Murilo Luiz [UNESP]
Perez-Riverol, Amilcar [UNESP]
Dos Santos-Pinto, José Roberto Aparecido [UNESP]
Fernandes, Luis Gustavo Romani
Lasa, Alexis Musacchio
Justo-Jacomini, Débora Laís [UNESP]
Palma, Mario Sergio [UNESP]
De Lima Zollner, Ricardo
Brochetto-Braga, Márcia Regina [UNESP]
author_role author
author2 Perez-Riverol, Amilcar [UNESP]
Dos Santos-Pinto, José Roberto Aparecido [UNESP]
Fernandes, Luis Gustavo Romani
Lasa, Alexis Musacchio
Justo-Jacomini, Débora Laís [UNESP]
Palma, Mario Sergio [UNESP]
De Lima Zollner, Ricardo
Brochetto-Braga, Márcia Regina [UNESP]
author2_role author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual Paulista (Unesp)
Universidade Estadual de Campinas (UNICAMP)
Center for Genetic Engineering and Biotechnology
dc.contributor.author.fl_str_mv Bazon, Murilo Luiz [UNESP]
Perez-Riverol, Amilcar [UNESP]
Dos Santos-Pinto, José Roberto Aparecido [UNESP]
Fernandes, Luis Gustavo Romani
Lasa, Alexis Musacchio
Justo-Jacomini, Débora Laís [UNESP]
Palma, Mario Sergio [UNESP]
De Lima Zollner, Ricardo
Brochetto-Braga, Márcia Regina [UNESP]
dc.subject.por.fl_str_mv Allergy
Antigen 5
Diagnosis
Heterologous expression
Polybia paulista
topic Allergy
Antigen 5
Diagnosis
Heterologous expression
Polybia paulista
description Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.
publishDate 2017
dc.date.none.fl_str_mv 2017-09-01
2018-12-11T17:14:26Z
2018-12-11T17:14:26Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.3390/toxins9090259
Toxins, v. 9, n. 9, 2017.
2072-6651
http://hdl.handle.net/11449/175114
10.3390/toxins9090259
2-s2.0-85028591746
2-s2.0-85028591746.pdf
2901888624506535
url http://dx.doi.org/10.3390/toxins9090259
http://hdl.handle.net/11449/175114
identifier_str_mv Toxins, v. 9, n. 9, 2017.
2072-6651
10.3390/toxins9090259
2-s2.0-85028591746
2-s2.0-85028591746.pdf
2901888624506535
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Toxins
0,955
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv Scopus
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
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