Partitioning and recovery of an elongation factor (1-γ) of Leishmania infantum chagasi expressed in E. coli M15 with simultaneous endotoxin removal using aqueous two-phase system
The 503-antigen, the elongation factor (1-γ) found in the amastigote phase of Leishmania infantum chagasi, could integrate either kits of specific diagnostic assays or vaccines for visceral leishmaniasis. However, protein purification expressed in Escherichia coli is driven by several steps, namely, clarification and multisteps chromatography. Additionally, the removal of lipopolysaccharide (LPS/endotoxin) which triggers an immune response in the human body is needed since LPS interacts with the proteins impairing the chromatography steps. Aqueous two-phase systems (ATPSs) based on polyethylene glycol (PEG) 1500 and salt (K2HPO4 or (NH4), ethanol/salt, and acetonitrile/D-glucose were used for partitioning of 503-antigen expressed in recombinant E. coli M15 with simultaneous LPSs removal. System based on 30% PEG 1500 and 10% potassium phosphate with 20% (w/w) unclarified cell homogenate achieved protein recovery of 116.96 ± 3.45% and purification factor of 1.55 ± 0.01. Endotoxin was removed with values as high as 99.90% for all systems with partial purification of 503-antigen, thus eliminating a clarification step in the partial purification protocol of this antigen.
