Non-glycosylated transferrin expressed in monocots

1. A method of producing a recombinant, non-glycosylated transferrin protein in rice plant seeds, said method comprising the steps of:

(a) transforming a rice plant cell with a chimeric gene comprising

(i) a promoter from a gene of a seed maturation-specific monocot plant storage protein;

(ii) a first DNA sequence, operably linked to said promoter, the first DNA sequence encoding a signal sequence targeting a polypeptide linked thereto to a protein storage body of a monocot plant seed cell; and

(iii) a second DNA sequence, linked in translation frame with the first DNA sequence, encoding a mammalian transferrin protein, wherein the first DNA sequence and the second DNA sequence together encode a fusion protein comprising the signal sequence and the transferrin protein;

(b) growing a rice plant from the transformed rice plant cell for a time sufficient to produce seeds containing the transferrin protein; and

(c) harvesting the seeds from the plant.

2. The method of claim 1 , wherein the second DNA sequence encoding the mammalian transferrin protein encodes a human transferrin protein having the amino acid sequence identified by SEQ ID NO. 3.

3. The method of claim 1 , wherein the second DNA sequence encoding the mammalian transferrin protein encodes a macaque transferrin protein having the amino acid sequence identified by SEQ ID NO. 27.

4. The method of claim 1 , wherein the first DNA sequence encoding a signal sequence targeting a polypeptide linked thereto to a protein storage body of a monocot plant seed cell encodes a rice glutelin signal sequence.

5. The method of claim 1 , wherein the promoter and signal sequence from the gene of a seed maturation-specific monocot plant storage protein encodes a glutelin (Gt1) promoter and signal sequence having the nucleic acid sequence identified by SEQ ID NO. 4.

6. A rice plant seed-derived composition, selected from the group consisting of a whole-seed composition, a flour composition, an extract composition and a malt composition, prepared from the harvested seeds obtained by the method of claim 1 .

7. The seed-derived composition of claim 6 , wherein the transferrin protein constitutes at least 0.1% of the dry weight of the seed-derived composition.

8. A seed-derived composition comprising a non-glycosylated transferrin protein, and at least one pharmaceutically acceptable excipient or nutrient, wherein the non-glycosylated transferrin protein is produced in a rice plant containing a nucleic acid sequence encoding the transferrin protein and is extracted from seed harvested from the rice plant.

9. A method for making a cell culture medium, said method comprising the steps of:

(a) obtaining a rice plant stably transformed with a chimeric gene having construct comprising

(i) a seed maturation-specific promoter;

(ii) a leader DNA sequence encoding a transit sequence targeting a linked polypeptide to a protein storage body of a rice plant seed cell; wherein said leader DNA sequence is operably linked to said promoter;

(iii) a protein-coding sequence encoding a transferrin protein;

(b) cultivating the transformed plant under seed-maturation conditions;

(c) harvesting the seeds from the cultivated plant;

(d) extracting the harvested seeds with an aqueous solution, thereby obtaining an extract of water soluble plant components comprising at least 3% by total protein weight of non-glycosylated transferrin protein;

(e) purifying the transferrin protein from the aqueous solution; and

(f) adding the purified transferrin protein to a cell culture medium.

10. The method of claim 1 , wherein the protein-coding sequence encodes human transferrin.

11. The method of claim 10 , wherein the human transferrin protein-coding sequence is the codon-optimized sequence identified by SEQ ID NO: 2.

12. The method of claim 10 , wherein the transformed rice plant further comprises a nucleic acid that encodes at least one transcription factor selected from the group consisting of Rice endosperm bZIP (Reb) (encoded by the sequence set forth as SEQ ID NO: 22), Opaque 2 (O2) (encoded by the sequence set forth as SEQ ID NO: 20) and prolamin box binding factor (PBF) (encoded by the sequence set forth as SEQ ID NO: 21).

13. The method of claim 12 , wherein the transcription factor is O2 and/or PBF.

14. A serum-free cell culture medium comprising an extract of rice seed comprising seed-expressed non-glycosylated transferrin protein.

15. The method of claim 1 , wherein the transferrin protein constitutes at least 0.01% seed weight of the harvested seeds.

16. The method of claim 1 , wherein the signal sequence in the fusion protein is located at the N-terminus.

17. A transformed rice plant produced according to the method of claim 1 .

18. A transformed rice seed produced according to the method of claim 1 .