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https://www.arca.fiocruz.br/handle/icict/14982
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
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
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.
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