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

Abstract

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.

Application of an aqueous two-phase systems strategy for the potential recovery of a recombinant protein from alfalfa (Medicago sativa)

“…Lane 6 corresponding to the bottom phase of PEG 600 system illustrates the absence of detectable proteins as expected from Bradford results. The absence of detectable bands in lane 7 (bottom phase of PEG 1450 system) is not corresponding with Bradford results for system 10 probably due to protein loss during precipitation step from PEG-rich systems, as previously documented [8,10]. In lane 8 the bottom phase of PEG 8000 system is showing the bands corresponding to the contaminants proteins added to the system.…”

“…Powder stocks were stored at −80 • C for further use. Protein extraction was performed as described before [10] using Tris-borate-ethylene diamine tetra acetic acid (EDTA) buffer (TBE) at a proportion of 1.0 g powdered alfalfa per 10 ml buffer. The slurry was stirred for 1.0 h, and then centrifuged at 12,000 × g for 10 min at room temperature (Centrifuge Galaxy16, VWR International, PA, USA).…”

“…In fact, potent systems have been developed to change the plant Nglycoforms to a desired or even superior form compared to the native mammalian N-glycoforms [3]. However, such experimental model represents a real challenge for downstream processing given the high concentration of contaminant proteins and the presence of the highly abundant protein Rubisco [10]. Therefore, the establishment of efficient primary recovery procedures for the recovery of recombinant proteins from transgenic crop is needed [9].…”

“…Previous reports have demonstrated the feasibility for recombinant human cytokines production in genetically modified plant cells [22,23]. The possibility of producing glycoforms of G-CSF in a commercially viable plant system has an enormous potential that can be explored, considering the increase in the biological activity of the molecule and the reduction in the production costs [10]. The artificial mixture formed by adding rhG-CSF to alfalfa green-tissue protein extract served as an example to simulate the presence of a recombinant protein.…”

Aqueous Two-Phase System Strategies for the Recovery of Proteins from Plants

“…protein Rubisco [ 12 ]. The following procedure can be used for the extraction of soluble protein from alfalfa leaves or any other green tissue with minimal or non-woody stems.…”