Purification of the recombinant rabies virus G glycoprotein (GPV) produced by Drosophila melanogaster S2 cells using immobilized metal ion affinity chromatography / Purificação da glicoproteina G (GPV) recombinante do virus da raiva produzida por celulas de Drosophila melanogaster S2 atraves de cromatografia de afinidade por ions metalicos imobilizados

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Rabies or hydrophobia is a viral infection that affects the central nervous system, occuring in animals and humans. The main proteins found in rabies virus that activates the immunological system are the N nucleoprotein (NPV) and the G glycoprotein, a transmembrane protein that forms the spikes of the virus and induces virus-neutralizing antibodies that protect against infection. This research aimed at the purification of the rabies virus glycoprotein containing a polyhistidine tag (GPV) from lisate and supernatant of Drosophila melanogaster Schneider 2 (S2AcGPV2) cells culture, transfected with the vector pAc 5.1/V5-His A containing the GPV gene, using immobilized metal íon affinity chromatography (IMAC). The aspects developed in this project were the agarose gel derivatization with the chelating agent iminodiacetic acid (IDA) and the evaluation of the agarose-IDA-Ni2+ gel seletivity, capacity and reproductibility at the GPV adsorption, regarding different buffer systems and different GPV?s desorption strategies (lowering the pH of the buffer or increasing the concentration of a competitive agent in the buffer). The seletivity in each buffer system was determined by performing SDS-PAGE electrophoresis on the chromatographic samples with the larger concentration of proteins, and the GPV quantification in these samples was determined by ELISA assays. The best results of purification were found when lisate was fed into an agarose-IDA-Ni2+ column equilibrated with 20 mM sodium phosphate buffer containing 500 mM sodium chloride and 2 mM imidazole pH 7,0. The washing step was proceeded with 20 mM sodium phosphate buffer containing 500 mM sodium chloride and 2 mM imidazole pH 6,0 and the elution proceeded by raising the imidazole concentration at the wash buffer to 200 e 500 mM. The results demonstrated the potencial of using IMAC to purify rabies virus G glycoprotein

ASSUNTO(S)

ions metalicos hydrophobia proteins proteinas - purificação metal ions cromatografia de afinidade quelantes affinity chromatography hidrofobia

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