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

摘要

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.