Clonagem e expressão da toxina TX3-1 do veneno da aranha phneutria nigriventer
Autor(a) principal: | |
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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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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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1816829592770445312 |