Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor

Detalhes bibliográficos
Autor(a) principal: Larentis, Ariane Leites
Data de Publicação: 2014
Outros Autores: Nicolau, Júlia Fabiana Monteiro Quintal, Esteves, Gabriela dos Santos, Vareschini, Daniel Tait, de Almeida, Fernanda Vicente Ribeiro, Reis, Mitermayer Galvão dos, Galler, Ricardo, Medeiros, Marco Alberto
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/14982
Resumo: Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Sergio Arouca National School of Public Health. ENSP, CESTEH. Rio de Janeiro, RJ, Brasil
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spelling Larentis, Ariane LeitesNicolau, Júlia Fabiana Monteiro QuintalEsteves, Gabriela dos SantosVareschini, Daniel Taitde Almeida, Fernanda Vicente RibeiroReis, Mitermayer Galvão dosGaller, RicardoMedeiros, Marco Alberto2016-07-19T17:41:37Z2016-07-19T17:41:37Z2014LARENTIS, A. L. et al. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Research Notes, v. 7, p. 671, 2014.1756-0500https://www.arca.fiocruz.br/handle/icict/1498210.1186/1756-0500-7-671engBioMed CentralEvaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactorinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Sergio Arouca National School of Public Health. ENSP, CESTEH. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, BrasilFundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biologia Molecular. Salvador, BA, BrasilFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Sergio Arouca National School of Public Health. ENSP, CESTEH. Rio de Janeiro, RJ, BrasilLeptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or non-existent sanitation infrastructure. Leptospirosis can kill, for its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira Immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz. Results: In this work, the process for expression of LigB (131-645aa) in E. coli BL21 (DE3)Star™/pAE was evaluated. No significant difference was found for the experiments at two different pre-induction temperatures (28°C and 37°C). Then, the strain was cultivated at 37°C until IPTG addition, followed by induction at 28°C, thereby reducing the overall process time. Under this condition, expression was assessed using central composite design for two variables: cell growth at which LigB (131-645aa) was induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1 mM to 1 mM IPTG). Both variables influenced cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Absind = 2.0 yielded higher cell concentrations and LigB (131-645aa) productivities. IPTG concentration had a negative effect and could be ten-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131-645aa) was associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28°C for 4 h was also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131-645aa) was purified by nickel affinity chromatography with 91% homogeneity. Conclusions: It was possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131-645aa) using experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis.LeptospiraLeptospirosisDiagnosisStatistical experimental designMicrobioreactorinfo: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/14982/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALLarentis AL Evaluation_of_pre-induction_tem....pdfLarentis AL Evaluation_of_pre-induction_tem....pdfapplication/pdf843106https://www.arca.fiocruz.br/bitstream/icict/14982/2/Larentis%20AL%20Evaluation_of_pre-induction_tem....pdf850087205ce92bac2431375828714df5MD52TEXTLarentis AL 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dc.title.pt_BR.fl_str_mv Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
title Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
spellingShingle Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
Larentis, Ariane Leites
Leptospira
Leptospirosis
Diagnosis
Statistical experimental design
Microbioreactor
title_short Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
title_full Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
title_fullStr Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
title_full_unstemmed Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
title_sort Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor
author Larentis, Ariane Leites
author_facet Larentis, Ariane Leites
Nicolau, Júlia Fabiana Monteiro Quintal
Esteves, Gabriela dos Santos
Vareschini, Daniel Tait
de Almeida, Fernanda Vicente Ribeiro
Reis, Mitermayer Galvão dos
Galler, Ricardo
Medeiros, Marco Alberto
author_role author
author2 Nicolau, Júlia Fabiana Monteiro Quintal
Esteves, Gabriela dos Santos
Vareschini, Daniel Tait
de Almeida, Fernanda Vicente Ribeiro
Reis, Mitermayer Galvão dos
Galler, Ricardo
Medeiros, Marco Alberto
author2_role author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Larentis, Ariane Leites
Nicolau, Júlia Fabiana Monteiro Quintal
Esteves, Gabriela dos Santos
Vareschini, Daniel Tait
de Almeida, Fernanda Vicente Ribeiro
Reis, Mitermayer Galvão dos
Galler, Ricardo
Medeiros, Marco Alberto
dc.subject.en.pt_BR.fl_str_mv Leptospira
Leptospirosis
Diagnosis
Statistical experimental design
Microbioreactor
topic Leptospira
Leptospirosis
Diagnosis
Statistical experimental design
Microbioreactor
description Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Sergio Arouca National School of Public Health. ENSP, CESTEH. Rio de Janeiro, RJ, Brasil
publishDate 2014
dc.date.issued.fl_str_mv 2014
dc.date.accessioned.fl_str_mv 2016-07-19T17:41:37Z
dc.date.available.fl_str_mv 2016-07-19T17:41:37Z
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 LARENTIS, A. L. et al. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Research Notes, v. 7, p. 671, 2014.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/14982
dc.identifier.issn.pt_BR.fl_str_mv 1756-0500
dc.identifier.doi.none.fl_str_mv 10.1186/1756-0500-7-671
identifier_str_mv LARENTIS, A. L. et al. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Research Notes, v. 7, p. 671, 2014.
1756-0500
10.1186/1756-0500-7-671
url https://www.arca.fiocruz.br/handle/icict/14982
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 BioMed Central
publisher.none.fl_str_mv BioMed Central
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/14982/1/license.txt
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