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.
abstract: BACKGROUND
  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 Abs(ind) = 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.
authors.affiliation: Fiocruz, Bio-Manguinhos, Vice Directory of Technological Development, Laboratory of Recombinant Technologies (LATER), Av, Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21,040-360, Brazil. arianelarentis@fiocruz.br.
authors.forename: Ariane Leites
authors.initials: AL
authors.lastname: Larentis
authors.forename: Júlia Fabiana Monteiro Quintal
authors.initials: JF
authors.lastname: Nicolau
authors.forename: Gabriela dos Santos
authors.initials: Gdos S
authors.lastname: Esteves
authors.forename: Daniel Tait
authors.initials: DT
authors.lastname: Vareschini
authors.forename: Fernanda Vicente Ribeiro
authors.initials: FV
authors.lastname: de Almeida
authors.forename: Mitermayer Galvão
authors.initials: MG
authors.lastname: dos Reis
authors.forename: Ricardo
authors.initials: R
authors.lastname: Galler
authors.forename: Marco Alberto
authors.initials: MA
authors.lastname: Medeiros
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D019149:Bioreactors; D002455:Cell Division; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D007919:Leptospira / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins