Abstract
BackgroundLeptospirosis 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.ResultsIn 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.ConclusionsIt 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.
Highlights
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
Expression of recombinant LigB to assess cell growth for induction (Absind) and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration Inoculum: 10 μL of the E. coli BL21 (DE3) StarTM/pAE/ LigB (131-645aa) stock was pre-inoculated in 10 mL Terrific Broth medium (TB) medium (23.6 g/L yeast extract, 11.8 g/L tryptone, 9.4 g/L K2HPO4, and 2.2 g/L KH2PO4, pH 7.2) enriched with 1% glucose, 0.4% glycerol and 100 μg/mL ampicillin for 16 h at 37°C and 200 rpm, in shaking 50 mL flasks
The bacteria were cultivated at 28°C and 37°C until absorbance reached 0.75, at which point IPTG was added to induce the expression of the recombinant protein LigB (131-645aa)
Summary
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 is a zoonotic disease caused by spirochetes of the genus Leptospira that occurs in tropical, subtropical and temperate climates. It is an increasing problem in cities, as people migrate from poor, rural areas to towns, where they often end up living in poor housing conditions with limited or no sanitation infrastructure, resulting in a change in the epidemiological profile of the disease. Leptospirosis has become a major public health issue, demanding increased investments in housing conditions and especially the development of rapid diagnosis and directed treatment methods. Phase leptospirosis is often missed or else is misdiagnosed and put down to some other cause of acute febrile disease because of its non-specific clinical presentation. It is ever more urgent that new strategies for diagnosis be developed and new diagnostic markers be identified, which can aid early case identification and timely administration of antibiotic therapy
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