Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer

Detalhes bibliográficos
Autor(a) principal: Andrea Cristina Gomes Pires
Data de Publicação: 2008
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da UFMG
Texto Completo: http://hdl.handle.net/1843/CMFC-7FJQUJ
Resumo: The toxin Tx3-1 purified from the spider venom Phoneutria nigriventer is a blocker of potassium channels with great pharmacological potential. However the purification of the toxin from Phoneutria nigriventer crude venom is a long and expensive process. The objective of this work was standardize techniques of molecular biology that would allow the expression and purification in large scale of functional recombinant Tx3-1.We cloned and expressed the toxin Tx3-1 tagged to the C-terminus of the maltose binding protein (MBP) in the cytoplasm of E. coli BL21. The hybrid protein purification was performed by affinity chromatography (amylase column). After purification, MBP-TX3-1 was cleaved by Factor Xa and recombinant Tx3-1 was purified by gel filtration using Superdex 75 column in FPLC system. Both, fusion protein MBP/Tx3-1 and recombinant Tx3-1 reacted with anti-Phoneutria nigriventer venom antibodies.The biological activity of recombinant toxin Tx3-1 was tested in biological assays and compared to native toxin biological activity. In experiments using rats cardiomyocytes, recombinant Tx3-1 toxin was able to increase the calcium transient in 20%, similarly to native toxin. Treatment of isolated rat hearts and atriums with recombinant Tx3-1, and native toxin, led to decrease in arrhythmia. These data suggest that the recombinant and native Tx3-1 probably present the same three-dimensional structure and function. These results open up the possibility of using the recombinant Tx3-1 to determine the molecular structure and action mechanisms of Tx3-1. Moreover, the recombinant protein enables the possibility of using the toxin Tx3-1 as a therapeutic drug for prevention and treatment of hearts diseases.
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spelling Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventerPHONEUTRIA NIGRIVENTERAranha VenenoBioquímicaThe toxin Tx3-1 purified from the spider venom Phoneutria nigriventer is a blocker of potassium channels with great pharmacological potential. However the purification of the toxin from Phoneutria nigriventer crude venom is a long and expensive process. The objective of this work was standardize techniques of molecular biology that would allow the expression and purification in large scale of functional recombinant Tx3-1.We cloned and expressed the toxin Tx3-1 tagged to the C-terminus of the maltose binding protein (MBP) in the cytoplasm of E. coli BL21. The hybrid protein purification was performed by affinity chromatography (amylase column). After purification, MBP-TX3-1 was cleaved by Factor Xa and recombinant Tx3-1 was purified by gel filtration using Superdex 75 column in FPLC system. Both, fusion protein MBP/Tx3-1 and recombinant Tx3-1 reacted with anti-Phoneutria nigriventer venom antibodies.The biological activity of recombinant toxin Tx3-1 was tested in biological assays and compared to native toxin biological activity. In experiments using rats cardiomyocytes, recombinant Tx3-1 toxin was able to increase the calcium transient in 20%, similarly to native toxin. Treatment of isolated rat hearts and atriums with recombinant Tx3-1, and native toxin, led to decrease in arrhythmia. These data suggest that the recombinant and native Tx3-1 probably present the same three-dimensional structure and function. These results open up the possibility of using the recombinant Tx3-1 to determine the molecular structure and action mechanisms of Tx3-1. Moreover, the recombinant protein enables the possibility of using the toxin Tx3-1 as a therapeutic drug for prevention and treatment of hearts diseases.A toxina Tx3-1 purificada do veneno da aranha Phoneutria nigriventer, é um bloqueador de canais de potássio, com grande potencial farmacológico. Porém a purificação dessa toxina a partir do veneno total da aranha Phoneutria nigriventer é um processo longo e dispendioso. O objetivo desse trabalho foi padronizar técnicas de biologia molecular que permitissem a expressão e purificação de grandes quantidades da toxina Tx3-1 recombinante com características funcionais semelhantes à da proteína nativa. Nós clonamos e expressamos a toxina Tx3-1 como um híbrido ligado a proteína ligadora de maltose (MBP) no citoplasma de E.coli BL21. Após purificação desta proteína híbrida por cromatografia de afinidade (coluna de amilose), a MBP-TX3-1 foi clivada por Fator Xa e a Tx3-1 recombinante foi purificada por gel filtração em coluna Superdex 75 em Sistema FPLC. Tanto a proteína de fusão MBP/Tx3-1 quanto a Tx3-1 recombinante reagiu com anticorpos antiveneno total da aranha Phoneutria nigriventer, indicando que a toxina foi clonada em fase de leitura correta. A atividade biológica da toxina Tx3-1 recombinante foi testada em ensaios biológicos e comparada com a atividade da toxina nativa. Semelhantemente à toxina nativa, em ensaios utilizando cardiomócitos de rato a toxina Tx3-1 recombinante foi capaz de aumentar a amplitude do cálcio transiente em 20%, quando comparada ao controle. Em ensaios com coração e átrios isolados de rato, a Tx3-1 recombinante apresentou efeito semelhante ao da Toxina Tx3-1 nativa, na redução do tempo de arritmia. Esses dados sugerem que a toxina Tx3-1 recombinante e a nativa apresentam a mesma estrutura tridimensional e abrem a possibilidade de utilizarmos a Tx3-1 recombinante para determinação da estrutura molecular da toxina e também para obter detalhes sobre o mecanismo molecular da ação da toxina. Além disso, a Tx3-1 recombinante viabiliza a possibilidade de utilização da toxina Tx3-1 como agente terapêutico para prevenção e tratamento de distúrbios cardíacos.Universidade Federal de Minas GeraisUFMGVania Ferreira PradoMarcus Vinicios GomezCarlos Delfin Chavez OlorteguiAndrea Cristina Gomes Pires2019-08-11T04:09:59Z2019-08-11T04:09:59Z2008-05-28info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/1843/CMFC-7FJQUJinfo:eu-repo/semantics/openAccessporreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMG2019-11-14T11:12:04Zoai:repositorio.ufmg.br:1843/CMFC-7FJQUJRepositório InstitucionalPUBhttps://repositorio.ufmg.br/oairepositorio@ufmg.bropendoar:2019-11-14T11:12:04Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
dc.title.none.fl_str_mv Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
title Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
spellingShingle Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
Andrea Cristina Gomes Pires
PHONEUTRIA NIGRIVENTER
Aranha Veneno
Bioquímica
title_short Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
title_full Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
title_fullStr Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
title_full_unstemmed Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
title_sort Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
author Andrea Cristina Gomes Pires
author_facet Andrea Cristina Gomes Pires
author_role author
dc.contributor.none.fl_str_mv Vania Ferreira Prado
Marcus Vinicios Gomez
Carlos Delfin Chavez Olortegui
dc.contributor.author.fl_str_mv Andrea Cristina Gomes Pires
dc.subject.por.fl_str_mv PHONEUTRIA NIGRIVENTER
Aranha Veneno
Bioquímica
topic PHONEUTRIA NIGRIVENTER
Aranha Veneno
Bioquímica
description The toxin Tx3-1 purified from the spider venom Phoneutria nigriventer is a blocker of potassium channels with great pharmacological potential. However the purification of the toxin from Phoneutria nigriventer crude venom is a long and expensive process. The objective of this work was standardize techniques of molecular biology that would allow the expression and purification in large scale of functional recombinant Tx3-1.We cloned and expressed the toxin Tx3-1 tagged to the C-terminus of the maltose binding protein (MBP) in the cytoplasm of E. coli BL21. The hybrid protein purification was performed by affinity chromatography (amylase column). After purification, MBP-TX3-1 was cleaved by Factor Xa and recombinant Tx3-1 was purified by gel filtration using Superdex 75 column in FPLC system. Both, fusion protein MBP/Tx3-1 and recombinant Tx3-1 reacted with anti-Phoneutria nigriventer venom antibodies.The biological activity of recombinant toxin Tx3-1 was tested in biological assays and compared to native toxin biological activity. In experiments using rats cardiomyocytes, recombinant Tx3-1 toxin was able to increase the calcium transient in 20%, similarly to native toxin. Treatment of isolated rat hearts and atriums with recombinant Tx3-1, and native toxin, led to decrease in arrhythmia. These data suggest that the recombinant and native Tx3-1 probably present the same three-dimensional structure and function. These results open up the possibility of using the recombinant Tx3-1 to determine the molecular structure and action mechanisms of Tx3-1. Moreover, the recombinant protein enables the possibility of using the toxin Tx3-1 as a therapeutic drug for prevention and treatment of hearts diseases.
publishDate 2008
dc.date.none.fl_str_mv 2008-05-28
2019-08-11T04:09:59Z
2019-08-11T04:09:59Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/1843/CMFC-7FJQUJ
url http://hdl.handle.net/1843/CMFC-7FJQUJ
dc.language.iso.fl_str_mv por
language por
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.publisher.none.fl_str_mv Universidade Federal de Minas Gerais
UFMG
publisher.none.fl_str_mv Universidade Federal de Minas Gerais
UFMG
dc.source.none.fl_str_mv reponame:Repositório Institucional da UFMG
instname:Universidade Federal de Minas Gerais (UFMG)
instacron:UFMG
instname_str Universidade Federal de Minas Gerais (UFMG)
instacron_str UFMG
institution UFMG
reponame_str Repositório Institucional da UFMG
collection Repositório Institucional da UFMG
repository.name.fl_str_mv Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)
repository.mail.fl_str_mv repositorio@ufmg.br
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