Method for recovering plant-derived proteins
Methods of recovering plant-derived proteins or suprastructure proteins, are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized proteins, or suprastructure proteins, loosening the cell wall to produce a plant or plant matter having a loosened cell wall, thus allowing the release of apoplastic content through the cell wall to produce an apoplastic content fraction from the plant or plant matter, and recovering the apoplastic content fraction. The apoplastic content fraction comprises plant-derived proteins or suprastructure proteins.
1. A method of recovering proteins, protein suprastructures or virus-like particles (VLPs) from plant or plant matter, comprising:
a. obtaining the plant or plant matter comprising apoplast-localized proteins, apoplast-localized protein suprastructures or apoplast-localized VLPs, the apoplast-localized protein suprastructures having a molecular weight from about 75 to about 1500 kDa;
b. treating the plant or plant matter with a composition comprising from about 25 to about 250 mM ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA) to loosen the cell wall to produce a plant or plant matter having a loosened cell wall, thereby releasing the apoplast-localized protein, apoplast-localized protein suprastructures, or apoplast-localized VLPs to produce a plant incubation mixture, separating the plant incubation mixture to produce a plant cell fraction and an apoplastic fraction; and
c. recovering the proteins, protein suprastructures, or the VLPs from the apoplastic fraction.
2. The method of claim 1 wherein the composition comprises ethylenediaminetetraacetic acid (EDTA).
3. The method of claim 1 wherein in the step of obtaining (step a), the plant is transformed with a nucleic acid sequence encoding the protein, protein suprastructures, or VLPs, the protein, protein suprastructures, or VLPs being selected from the group of a protein, a protein rosette, a protein complex, a proteasome, a metabolon, a transcription complex, a recombination complex, a photosynthetic complex, a membrane transport complex, a nuclear pore complex, a protein nanoparticle, a glycoprotein, an antibody, a polyclonal antibody, a monoclonal antibody, a single chain monoclonal antibody, a virus like particle, a viral envelope protein, a viral structural protein, a viral capsid protein, and a viral coat protein, a chimeric protein, a chimeric protein complex, a chimeric protein nanoparticle, a chimeric glycoprotein, a chimeric antibody, a chimeric monoclonal antibody, a chimeric single chain monoclonal antibody, a chimeric hemagglutinin, and then the transformed plant or plant matter is harvested.
4. The method of claim 3 , wherein the nucleic acid is introduced into the plant in a transient manner.
5. The method of claim 3 , wherein, the nucleic acid is stably integrated within a genome of the plant.
6. The method of claim 1 wherein in the step of obtaining (step a)[ ], the plant is grown and the plant or plant matter is harvested.
7. The method of claim 3 wherein the nucleic acid encodes a monoclonal antibody or an influenza hemagglutinin.
8. The method of claim 1 wherein the plant-derived proteins, or protein suprastructures do not include neuraminidase or M protein.
9. The method of claim 1 wherein the plant matter is selected from the group of leaves, and cultured plant cells.
10. The method of claim 1 further comprising a step of d) purifying the proteins, protein suprastructures or VLPs, from the apoplastic fraction.
11. The method of claim 10 , wherein the step of d) purifying comprises filtering the apoplastic fraction using depth filtration to produce a clarified extract, followed by chromatography of the clarified extract using size exclusion chromatography, cation exchange resin or affinity chromatography, or a combination thereof.
12. The method of claim 1 , wherein the step of treating (step b), separating is performed by centrifugation, depth filtration, or a combination thereof.
13. The method of claim 1 , wherein the composition is introduced into the plant or plant matter by infiltration under partial vacuum.
14. The method of claim 1 , wherein the step of treating (step b) further comprises sonication.
15. The method of claim 1 , wherein in the step of treating (step b), the composition further comprises an enzyme mixture.
16. The method of claim 15 , wherein the enzyme mixture is introduced into the plant or plant matter by enzymatic infiltration.
17. The method of claim 16 , wherein the enzymatic infiltration is selected from the group of vacuum or pressure infiltration.
18. The method of claim 15 , wherein the enzyme mixture comprises one or more than one pectinase, one or more than one cellulase, or one or more than one pectinase and one or more than one cellulase.
19. The method of claim 15 , wherein the enzyme mixture does not include one or more of a lipase, a protease or a pectinase.