Recovery of recombinant proteins CFP10 and ESAT6 from Escherichia coli inclusion bodies for tuberculosis diagnosis: a statistical optimization approach

摘要

Tuberculosis (TB) is among the top ten causes of mortality worldwide and has high prevalence in developing countries. The dissemination of efficient and low-cost diagnosis tools able to identify its latent form, e.g. the delayed hypersensitivity reaction, is of great importance to accomplish the target of TB eradication. Recent studies have shown the potential of specific Mycobacterium tuberculosis immunodominant antigens, CFP10 and ESAT6, as substitutes of tuberculin skin test. The purpose of this study was to optimize the recovery and purification of recombinant CFP10 and ESAT6 from Escherichia coli inclusion bodies using central composite design. The production of CFP10 and ESAT6 in bioreactor presented yields of 233 and 121?mg?L?1, respectively, after extraction under optimized conditions: biomass concentration of 15?g per 150?mL of sonication buffer, using 12 cycles of disruption and 7 cycles of solubilization, followed by affinity chromatography purification and removal of endotoxins by the micellar method. The identity of the antigens was confirmed by mass spectrometry and their immunoreactivity after recovery and purification was confirmed by Western blot. These results demonstrate the suitability of the preparation methods in the development of a TB diagnosis kit with potential to be diffused in high TB burden countries.