Chemical extraction and direct adsorptive purification of recombinant human antigen Ro52

The antigenic protein Ro52 was expressed in the E. coli system harboring a 6×His tag in the form of insoluble inclusion bodies. Direct chemical extraction of the product using 6–8 M urea proved to be effective. Furthermore, the tagged protein was recovered by direct adsorption on Ni2+-loaded commercial adsorbents derivatized with iminodiacetic acid. Screening experiments in small packed columns revealed that selective binding and elution were possible using a denaturing buffer at pH 4.5. The hydrodynamic evaluation of scaled-up fluidized systems showed values for the φ (dynamic zone) parameter in the range 0.95–1.00 for fluidization in buffer and in the range 0.70–0.85 for the biomass-containing feedstock. Removal of macromolecular DNA released by the disrupted biomass was mandatory. Under optimized process conditions good recovery (60–70%) was achieved and a highly purified (95%) product obtained. The purified Ro52 retained its immunoreactivity against sera of patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome-related disorders. The production and application of new recombinant antigens may contribute to increasing the sensitivity and specificity of the detection of anti-Ro antibodies in these autoimmune diseases.