Aqueous two-phase systems for purification of recombinant proteins expressed in plants.

MOZGOVOJ, M. V., OSTACHUK, A., WOLMAN, F. J., DUS SANTOS, M. J., WIDOROWITZ, A., CASCONE, O.

Mar del Plata, Buenos Aires, Argentina

Congreso; SAIB 2007, XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2007

Sociedad Argentina de Investigiación en Bioquímica y Biología Molecular

Bovine viral diarrhea virus (BVDV) and bovine rotavirus (BRV) cause significant economic losses in Argentina. Proteins E2T and VP8 from BVDV and BRV are being expressed in alphalpha to develop vaccines. In this way, aqueous two-phase partition systems (ATPS) are eligible to purify them because of their low cost and easy scale-up. In these systems, experiments have to be performed to find differential partition (the protein of interest is in one phase while the contaminants partition mainly in the other phase). The effect of PEG molecular weight, pH and added salts in PEG/phosphate ATPS was assessed. In the case of E2T, the higher the PEG molecular weight, the higher was the partition constant (18 for PEG 4000). Salts added and pH variations failed to change significantly the partition behavior of the protein. In the case of VP8, the partition constant was higher than 11 for PEG 600/phosphate and PEG 1500/phosphate ATPS. The addition of 0.5 M NaCl to PEG 1500/phosphate ATPS increased the partition constant by 13 % while the partition constant of the proteins in the PEG 600/phosphate ATPS remained essentially unchanged under the same conditions. pH variation did not bring about any effect on the VP8 partition. Results obtained allow us to establish the conditions for recuperation of both protein with high yield, low cost and the possibility to carry out the scale-up easily.