Method of producing a lunasin polypeptide in plants
A method of producing a lunasin polypeptide in a plant includes expressing a fusion protein including the lunasin polypeptide in the plant and cleaving the lunasin polypeptide from the fusion protein.
1. A method of producing a lunasin polypeptide in a plant, said method comprising:
introducing an expression vector comprising a polynucleotide encoding a fusion protein into a plant cell, said fusion protein comprising
the lunasin polypeptide;
a green fluorescent protein; and
a site-specific protease cleavage site; and
expressing the fusion protein within the plant, wherein said fusion protein is not toxic to the plant cell.
2. The method of claim 1 , wherein the expression vector is a transient expression vector and the plant cell is a non-dividing plant cell.
3. The method of claim 1 , wherein the expression vector is a tobacco mosaic virus (TMV)-based DNA plasmid.
4. The method of claim 1 , wherein the plant cell is a Nicotiana benthamiana cell.
5. The method of claim 1 further comprising isolating the fusion protein.
6. The method of claim 5 further comprising cleaving the lunasin polypeptide from the fusion protein.
7. The method of claim 5 , and further comprising cleaving a lunasin polypeptide-containing fusion protein from the remainder of the fusion protein expressed within the plant.
8. The method of claim 7 , wherein the lunasin polypeptide-containing fusion protein further comprises a polypeptide targeting signal for directing the lunasin polypeptide to a target cell.
9. The method of claim 1 , wherein the lunasin polypeptide is produced in amounts of about >100 mg/kg fresh weight plant tissue.
10. A method of producing a lunasin polypeptide in a plant, said method comprising:
providing an expression vector comprising a polynucleotide encoding a fusion protein including:
the lunasin polypeptide;
a green fluorescent protein; and
a site-specific protease cleavage site;
introducing the expression vector into a plant cell;
isolating the fusion protein; and
using a protease of the site-specific protease cleavage site to obtain the lunasin polypeptide or a desired lunasin polypeptide-containing fusion protein, wherein said fusion protein is not toxic to the plant cell.
11. The method of claim 10 , wherein the lunasin polypeptide is produced in amounts of about >100 mg/kg fresh weight plant tissue.
12. The method of claim 10 , wherein the desired lunasin polypeptide-containing fusion protein further comprises a polypeptide targeting signal for directing the lunasin polypeptide to a target cell.
13. The method of claim 10 , wherein the expression vector is a transient expression vector and the plant cell is a non-dividing plant cell.
14. The method of claim 10 , wherein the expression vector is a tobacco mosaic virus (TMV)-based DNA plasmid.
15. The method of claim 10 , wherein the plant cell is a Nicotiana benthamiana cell.