Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris

Detalhes bibliográficos
Autor(a) principal: Vieira, Saulo Martins
Data de Publicação: 2017
Outros Autores: Rocha, Surza Lucia Gonçalves da, Ferreira, Ana Gisele da Costa Neves, Almeida, Rodrigo Volcan, Perales, Jonas
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/24835
Resumo: Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq. Brasília, DF, Brasil.
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spelling Vieira, Saulo MartinsRocha, Surza Lucia Gonçalves daFerreira, Ana Gisele da Costa NevesAlmeida, Rodrigo VolcanPerales, Jonas2018-02-15T14:40:03Z2018-02-15T14:40:03Z2017VIEIRA, Saulo Martins; et al. Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris. PLoS Negl Trop Dis., v.11, n.7, e0005829, 20p, July 2017.1935-2727https://www.arca.fiocruz.br/handle/icict/2483510.1371/journal.pntd.00058291935-2735engPublic Library of SciencePichia pastorisproteína antimiotóxicaDM64Pichia pastorisantimyotoxic proteinDM64Heterologous expression of the antimyotoxic protein DM64 in Pichia pastorisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq. Brasília, DF, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Química. Departamento de Bioquímica. Laboratório de Microbiologia Molecular e Proteínas. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq. Brasília, DF, Brasil.Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/24835/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALsaulo_vieira_etal_IOC_2017.pdfsaulo_vieira_etal_IOC_2017.pdfapplication/pdf16125317https://www.arca.fiocruz.br/bitstream/icict/24835/2/saulo_vieira_etal_IOC_2017.pdf1bf58e7c1af64a73630a068b44f746caMD52TEXTsaulo_vieira_etal_IOC_2017.pdf.txtsaulo_vieira_etal_IOC_2017.pdf.txtExtracted 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dc.title.pt_BR.fl_str_mv Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
title Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
spellingShingle Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
Vieira, Saulo Martins
Pichia pastoris
proteína antimiotóxica
DM64
Pichia pastoris
antimyotoxic protein
DM64
title_short Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
title_full Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
title_fullStr Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
title_full_unstemmed Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
title_sort Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
author Vieira, Saulo Martins
author_facet Vieira, Saulo Martins
Rocha, Surza Lucia Gonçalves da
Ferreira, Ana Gisele da Costa Neves
Almeida, Rodrigo Volcan
Perales, Jonas
author_role author
author2 Rocha, Surza Lucia Gonçalves da
Ferreira, Ana Gisele da Costa Neves
Almeida, Rodrigo Volcan
Perales, Jonas
author2_role author
author
author
author
dc.contributor.author.fl_str_mv Vieira, Saulo Martins
Rocha, Surza Lucia Gonçalves da
Ferreira, Ana Gisele da Costa Neves
Almeida, Rodrigo Volcan
Perales, Jonas
dc.subject.other.pt_BR.fl_str_mv Pichia pastoris
proteína antimiotóxica
DM64
topic Pichia pastoris
proteína antimiotóxica
DM64
Pichia pastoris
antimyotoxic protein
DM64
dc.subject.en.pt_BR.fl_str_mv Pichia pastoris
antimyotoxic protein
DM64
description Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq. Brasília, DF, Brasil.
publishDate 2017
dc.date.issued.fl_str_mv 2017
dc.date.accessioned.fl_str_mv 2018-02-15T14:40:03Z
dc.date.available.fl_str_mv 2018-02-15T14:40:03Z
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.citation.fl_str_mv VIEIRA, Saulo Martins; et al. Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris. PLoS Negl Trop Dis., v.11, n.7, e0005829, 20p, July 2017.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/24835
dc.identifier.issn.pt_BR.fl_str_mv 1935-2727
dc.identifier.doi.none.fl_str_mv 10.1371/journal.pntd.0005829
dc.identifier.eissn.none.fl_str_mv 1935-2735
identifier_str_mv VIEIRA, Saulo Martins; et al. Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris. PLoS Negl Trop Dis., v.11, n.7, e0005829, 20p, July 2017.
1935-2727
10.1371/journal.pntd.0005829
1935-2735
url https://www.arca.fiocruz.br/handle/icict/24835
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:Repositório Institucional da FIOCRUZ (ARCA)
instname:Fundação Oswaldo Cruz (FIOCRUZ)
instacron:FIOCRUZ
instname_str Fundação Oswaldo Cruz (FIOCRUZ)
instacron_str FIOCRUZ
institution FIOCRUZ
reponame_str Repositório Institucional da FIOCRUZ (ARCA)
collection Repositório Institucional da FIOCRUZ (ARCA)
bitstream.url.fl_str_mv https://www.arca.fiocruz.br/bitstream/icict/24835/1/license.txt
https://www.arca.fiocruz.br/bitstream/icict/24835/2/saulo_vieira_etal_IOC_2017.pdf
https://www.arca.fiocruz.br/bitstream/icict/24835/3/saulo_vieira_etal_IOC_2017.pdf.txt
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repository.name.fl_str_mv Repositório Institucional da FIOCRUZ (ARCA) - Fundação Oswaldo Cruz (FIOCRUZ)
repository.mail.fl_str_mv repositorio.arca@fiocruz.br
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