Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics
Autor(a) principal: | |
---|---|
Data de Publicação: | 2020 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1007/s13205-020-02202-8 http://hdl.handle.net/11449/200339 |
Resumo: | Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate–phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy. |
id |
UNSP_35ab19ab460d11d3bc23fa2f16c3179c |
---|---|
oai_identifier_str |
oai:repositorio.unesp.br:11449/200339 |
network_acronym_str |
UNSP |
network_name_str |
Repositório Institucional da UNESP |
repository_id_str |
2946 |
spelling |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnosticsMolecular diagnosticsPolybia paulistaRecombinant phospholipase A1RecoverySolubilizationVenom allergyPhospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate–phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC)Center for Genetic Engineering and Biotechnology. Biomedical Research Division System Biology Department, Ave. 31, e/ 158 and 190, Cubanacan, Playa, P.O. Box 6162Laboratory of Translational Immunology School of Medical Sciences University of Campinas (UNICAMP), CampinasArthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela VistaVenoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC)Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela VistaVenoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780FAPESP: # 2014/13936-7Universidade Estadual Paulista (Unesp)System Biology DepartmentUniversidade Estadual de Campinas (UNICAMP)Perez-Riverol, Amilcar [UNESP]Musacchio-Lasa, AlexisFernandes, Luis Gustavo Romanidos Santos-Pinto, Jose Roberto Aparecido [UNESP]Esteves, Franciele Grego [UNESP]Bazon, Murilo Luiz [UNESP]Zollner, Ricardo de LimaPalma, Mario Sergio [UNESP]Brochetto-Braga, Márcia Regina [UNESP]2020-12-12T02:04:03Z2020-12-12T02:04:03Z2020-05-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1007/s13205-020-02202-83 Biotech, v. 10, n. 5, 2020.2190-57382190-572Xhttp://hdl.handle.net/11449/20033910.1007/s13205-020-02202-82-s2.0-85083969120Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPeng3 Biotechinfo:eu-repo/semantics/openAccess2024-04-11T14:57:11Zoai:repositorio.unesp.br:11449/200339Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-04-11T14:57:11Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
title |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
spellingShingle |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics Perez-Riverol, Amilcar [UNESP] Molecular diagnostics Polybia paulista Recombinant phospholipase A1 Recovery Solubilization Venom allergy |
title_short |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
title_full |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
title_fullStr |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
title_full_unstemmed |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
title_sort |
Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics |
author |
Perez-Riverol, Amilcar [UNESP] |
author_facet |
Perez-Riverol, Amilcar [UNESP] Musacchio-Lasa, Alexis Fernandes, Luis Gustavo Romani dos Santos-Pinto, Jose Roberto Aparecido [UNESP] Esteves, Franciele Grego [UNESP] Bazon, Murilo Luiz [UNESP] Zollner, Ricardo de Lima Palma, Mario Sergio [UNESP] Brochetto-Braga, Márcia Regina [UNESP] |
author_role |
author |
author2 |
Musacchio-Lasa, Alexis Fernandes, Luis Gustavo Romani dos Santos-Pinto, Jose Roberto Aparecido [UNESP] Esteves, Franciele Grego [UNESP] Bazon, Murilo Luiz [UNESP] Zollner, Ricardo de Lima Palma, Mario Sergio [UNESP] 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) System Biology Department Universidade Estadual de Campinas (UNICAMP) |
dc.contributor.author.fl_str_mv |
Perez-Riverol, Amilcar [UNESP] Musacchio-Lasa, Alexis Fernandes, Luis Gustavo Romani dos Santos-Pinto, Jose Roberto Aparecido [UNESP] Esteves, Franciele Grego [UNESP] Bazon, Murilo Luiz [UNESP] Zollner, Ricardo de Lima Palma, Mario Sergio [UNESP] Brochetto-Braga, Márcia Regina [UNESP] |
dc.subject.por.fl_str_mv |
Molecular diagnostics Polybia paulista Recombinant phospholipase A1 Recovery Solubilization Venom allergy |
topic |
Molecular diagnostics Polybia paulista Recombinant phospholipase A1 Recovery Solubilization Venom allergy |
description |
Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate–phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-12-12T02:04:03Z 2020-12-12T02:04:03Z 2020-05-01 |
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/10.1007/s13205-020-02202-8 3 Biotech, v. 10, n. 5, 2020. 2190-5738 2190-572X http://hdl.handle.net/11449/200339 10.1007/s13205-020-02202-8 2-s2.0-85083969120 |
url |
http://dx.doi.org/10.1007/s13205-020-02202-8 http://hdl.handle.net/11449/200339 |
identifier_str_mv |
3 Biotech, v. 10, n. 5, 2020. 2190-5738 2190-572X 10.1007/s13205-020-02202-8 2-s2.0-85083969120 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
3 Biotech |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
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) |
repository.mail.fl_str_mv |
|
_version_ |
1797789793425293312 |