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.
Saccodossi
Natalia
N
Cátedra de Inmunología, IDEHU-CONICET-UBA, Buenos Aires, Argentina.
Cabrera
Rosa B
RB
Oliver
Fernando
F
Gelmi
Lucila
L
Magglio
Daniel G
DG
Fernández-Lahore
Hector M
HM
Leoni
Juliana
J
Stumpo
Rita R
RR
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
D001324:Autoantigens; D017931:DNA Primers; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D013096:Spermine; D000099207:SS-A Antigen