(556b) Application of ATPS Strategies for the Potential Recovery of Recombinant Proteins From of Alfalfa Extracts

In order to facilitate the general acceptance of plants as bioreactors, the establishment of efficient downstream operations is critical. It has been proposed recently that a better understanding of the properties of the contaminant proteins could benefit downstream processing design and operation (1,2). However, the lack of efficient initial concentration and separation procedures affect the generic acceptance of plants as economically viable systems. In this context the use of aqueous two-phase systems (ATPS) can provide strategies to facilitate the adoption of plants as a base for bioprocesses (2).

In the present study, ATPS was explored as a potential technique for the primary recovery of a model human recombinant protein (rhGM-CSF, granulocyte-macrophage colony stimulating factor) from alfalfa green tissue. The characterization of the partitioning behavior of the potential contaminants and the model recombinant protein was studied as the first step for the establishment of a potential downstream strategy for the recovery of recombinant proteins expressed in green tissues.

Water-soluble proteins extracted from alfalfa (Medicago sativa), were considered as the potential contaminants for the design of a recovery strategy of rhGM-CSF. PEG-phosphate ATPS were designed for the characterization of partitioning behavior of alfalfa proteins using 5 molecular weights of polyethylene glycol (600, 1000, 1450, 3350 and 8000 g/mol) and 4 different tie line lengths (TLL). Sample was added as 10% (w/w) of the total system weight. All the experiments were performed at 22° C and pH 7.

The evaluation of the effect of system parameters provided the conditions under which the contaminant proteins from alfalfa extracts and rhGM-CSF can be potentially concentrated in opposite phases. The results showed that the use of low molecular weight of PEG (600, 1000 and 1450) allowed the recovery of most of the alfalfa proteins in the upper phase. According with these preliminary results, five PEG/phosphate systems were selected for the characterization of rhGM-CSF partitioning behavior, resulting a marked preference of the model protein for the lower phase. A potential recovery strategy for this model protein from the lower phase of PEG/phosphate systems was established using PEG 1000, 1450 and 3350.

References

1. Characterization of green-tissue protein extract from alfalfa (Medicago sativa) exploiting a 3D technique. Aguilar, Oscar, Glatz, Charles E. and Rito-Palomares, Marco. Sent for publication.

2. Processing of soybean (Glycine max) extracts in aqueous two-phase systems as a first step for the potential recovery of recombinant proteins. Aguilar, Oscar and Rito-Palomares, Marco. 2008, Journal of Chemical Technology and Biotechnology, Vol. 83, pp. 286?293.