title: 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.
authors.affiliation: Cátedra de Inmunología, IDEHU-CONICET-UBA, Buenos Aires, Argentina.
authors.forename: Natalia
authors.initials: N
authors.lastname: Saccodossi
authors.forename: Rosa B
authors.initials: RB
authors.lastname: Cabrera
authors.forename: Fernando
authors.initials: F
authors.lastname: Oliver
authors.forename: Lucila
authors.initials: L
authors.lastname: Gelmi
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Magglio
authors.forename: Hector M
authors.initials: HM
authors.lastname: Fernández-Lahore
authors.forename: Juliana
authors.initials: J
authors.lastname: Leoni
authors.forename: Rita R
authors.initials: RR
authors.lastname: Stumpo
mesh_terms: D000327:Adsorption; D000821:Animal Feed; D001324:Autoantigens / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D011994:Recombinant Proteins / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D013096:Spermine; D000099207:SS-A Antigen
chemical_list: D001324:Autoantigens; D017931:DNA Primers; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D013096:Spermine; D000099207:SS-A Antigen