Methods for the production of cytoprotective asialo-erythropoietin in plants and its purification from plant tissues

A method for the high-level production and purification of asialoerythropoietin (asialo-rhuEPO) in a plant or plant cell comprising:

• (a) obtaining a transformed tobacco plant or transformed tobacco plant cell, wherein the transformed plant or transformed plant cell comprises a first promoter that drives expression in a plant cell operably linked to a first polynucleotide encoding a human erythropoietin (EPO) fusion protein and a second promoter that drives expression in a plant cell operably linked to a second polynucleotide encoding a β1,4-galactosyltransferase;
• wherein the human EPO fusion protein is encoded by a nucleic acid sequence selected from the group consisting of:
• (1) the nucleic acid sequence set forth in SEQ ID NO:
• 3;
• and
• (2) a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:
• 4;
• and
• wherein the second polynucleotide comprises a nucleic acid sequence selected from the group consisting of:
• (1) the nucleic acid sequence set forth in SEQ ID NO:
• 10;
• and
• (2) a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:
• 11;
• (b) making an aqueous extract of plant tissue, wherein the plant tissue comprises asialo-rhuEPO;
• (c) removing chlorophyll and/or RuBisCO protein from the aqueous extract via ammonium sulfate precipitation;
• (d) binding the asialo-rhuEPO in the aqueous extract to an immune affinity column, wherein the immune affinity column is a rabbit anti-EPO polyclonal antibody column;
• and
• (e) eluting the bound asialo-rhuEPO from the immune affinity column;
• wherein at least 100 ng of asialo-rhuEPO per mg of total soluble protein is recovered;
• and
• wherein the asialo-rhuEPO comprises terminal β1,4-galactose residues.