Chemical extraction and direct adsorptive purification of recombinant human antigen Ro52.

NATALIA SACCODOSSI; ROSA B. CABRERA; FERNANDO OLIVER; LUCILA GELMI; DANIEL G. MAGLIO; HÉCTOR M. FERNÁNDEZ-LAHORE; JULIANA LEONI; RITA R. STUMPO

JOURNAL OF SEPARATION SCIENCE - (Online)

Wiley-VCH Verlag GmbH & Co

Año: 2004 vol. 27 p. 589 - 594

1615-9314

The antigenic protein Ro52 was expressed in the E. coli system harboring a 66Histag in the form of insoluble inclusion bodies. Direct chemical extraction of the productusing 6–8 M urea proved to be effective. Furthermore, the tagged protein was recoveredby direct adsorption on Ni2+-loaded commercial adsorbents derivatized with iminodiaceticacid. Screening experiments in small packed columns revealed that selectivebinding and elution were possible using a denaturing buffer at pH 4.5. The hydrodynamicevaluation of scaled-up fluidized systems showed values for the u (dynamiczone) parameter in the range 0.95–1.00 for fluidization in buffer and in the range0.70–0.85 for the biomass-containing feedstock. Removal of macromolecular DNAreleased by the disrupted biomass was mandatory. Under optimized process conditionsgood recovery (60–70%) was achieved and a highly purified (95%) productobtained. The purified Ro52 retained its immunoreactivity against sera of patientswith systemic lupus erythematosus (SLE) and Sjögren´s syndrome-related disorders.The production and application of new recombinant antigens may contribute toincreasing the sensitivity and specificity of the detection of anti-Ro antibodies in theseautoimmune diseases.