Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method.

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123-128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 µg of polygalacturonic acid, and 45 µg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.

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 x 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 phi (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.