Characterization of green‐tissue protein extract from alfalfa (Medicago sativa) exploiting a 3‐D technique

There is a growing interest of pharmaceutical companies for plant‐based production systems. To facilitate the general acceptance of plants as bioreactors, the establishment of efficient downstream operations is critical. It has been proposed that a better understanding of the properties of the contaminant proteins can benefit downstream processing design and operation. The coupled application of 2‐DE with aqueous two‐phase partitioning has been suggested as a practical 3‐D method to characterize potential contaminant proteins from plant extracts. The application of this novel 3‐D approach to a complex protein extract from alfalfa (Medicago sativa) containing a model recombinant protein (human granulocyte colony stimulating factor (hG‐CSF)) resulted in the quantification of 55 protein spots. The 3‐D properties (M_r, pI, and K_p) obtained for 17 proteins comprising 69% of the alfalfa proteins, allowed the proposal of a prefractionation step as well as the identification of the target molecule (rG‐CSF) from bulk of alfalfa proteins. The information obtained from this experimental approach was useful for the identification of the potential contaminant proteins that will occur in alfalfa when this plant is used as a host for recombinant proteins. Additionally, this method will assist in the design of adequate purification strategies for recombinant proteins expressed in alfalfa green tissue.