Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/14982
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
Authors: 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
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
Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil
Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil
Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil
Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil
Fundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biologia Molecular. Salvador, BA, Brasil
Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratory of Recombinant Technologies. LATER. Rio de Janeiro, RJ, Brasil
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
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.
Keywords: Leptospira
Leptospirosis
Diagnosis
Statistical experimental design
Microbioreactor
Issue Date: 2014
Publisher: BioMed Central
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.
DOI: 10.1186/1756-0500-7-671
ISSN: 1756-0500
Copyright: open access
Appears in Collections:Biomanguinhos - Artigos de Periódicos
BA - IGM - Artigos de Periódicos

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