Author | Larentis, Ariane Leites | |
Author | Nicolau, Júlia Fabiana Monteiro Quintal | |
Author | Esteves, Gabriela dos Santos | |
Author | Vareschini, Daniel Tait | |
Author | de Almeida, Fernanda Vicente Ribeiro | |
Author | Reis, Mitermayer Galvão dos | |
Author | Galler, Ricardo | |
Author | Medeiros, Marco Alberto | |
Access date | 2016-07-19T17:41:37Z | |
Available date | 2016-07-19T17:41:37Z | |
Document date | 2014 | |
Citation | 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. | pt_BR |
ISSN | 1756-0500 | pt_BR |
URI | https://www.arca.fiocruz.br/handle/icict/14982 | |
Language | eng | pt_BR |
Publisher | BioMed Central | pt_BR |
Rights | open access | pt_BR |
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 | pt_BR |
Type | Article | pt_BR |
DOI | 10.1186/1756-0500-7-671 | |
Abstract | Leptospirosis 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. | pt_BR |
Affilliation | 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 | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil | pt_BR |
Affilliation | Fundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biologia Molecular. Salvador, BA, Brasil | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil | pt_BR |
Affilliation | 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 | pt_BR |
Subject | Leptospira | pt_BR |
Subject | Leptospirosis | pt_BR |
Subject | Diagnosis | pt_BR |
Subject | Statistical experimental design | pt_BR |
Subject | Microbioreactor | pt_BR |