Chemical extraction and direct adsorptive purification of recombinant human antigen Ro52

Abstract

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.

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

“…The present results have practical implications during processing schemes where biomass chemical extraction coupled to direct sequestration is sought as a means for downstream processes integration [24].…”

Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant Streptokinase from E. coli inclusion bodies

“…1) for SK from E. coli inclusion bodies. This approach has so far been tried only with affinity-based matrices [3,4]. Here, for the first time we have established the utility of hydrophobic interaction based EBAC (HI-EBAC) procedure for achieving the same end, and could thereby achieve a purification yield of around 80% and a 3-fold increase in purity.…”

“…In the recent literature some reports on the use of affinity based EBAC in conjunction with chemical extraction [3,4,25,26] are present. In this study, however, a hydrophobic interaction based EBAC was attempted using phenyl-based Streamline matrix.…”

“…It involves the use of chaotropic concentrations of urea and EDTA containing buffers. In more recent reports, direct adsorption of the desired protein from the unclarified post-extraction solution (obtained after chemical extraction) using IMAC based EBA adsorbents have been described [3,4]. The use of IMAC based adsorbents, however, necessitate the expression of target protein with a histidine tag at the N-or C-terminal, which may not be desirable in case of injectible therapeutic proteins for human applications.…”