Expression of epidermal growth factor in plant seeds
The present invention provides a method of preparing epidermal growth factors in plants and transgenic plant seeds containing epidermal growth factors. The method provides an economical way to produce epidermal growth factors.
1. A method for the expression of an epidermal growth factor in plants said method comprising:
(a) introducing into a plant cell a chimeric nucleic acid sequence comprising:
(1) a first nucleic acid sequence capable of regulating the transcription in said plant cell of
(2) a second nucleic acid sequence, wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in frame to (ii) a nucleic acid sequence encoding said epidermal growth factor; and
(3) a third DNA sequence encoding a termination region functional in said plant cell; and
(b) growing said plant cell to produce said recombinant fusion polypeptide.
2. The method according to claim 1 further including separating the recombinant fusion polypeptide from cellular host cell components by selective partitioning into a lipid phase.
3. The method according to claim 1 further including separating the recombinant fusion polypeptide from cellular host components by selective partitioning into a lipid phase comprising oil bodies.
4. A method according to claim 1 wherein said oil body protein is an oleosin, caleosin or steroleosin.
5. A method according to claim 4 wherein said oleosin is SEQ.ID.NO.: 1 or SEQ.ID.NO.: 2, wherein said caleosin is encoded by SEQ.ID.NO.:3 or SEQ.ID.NO.: 4 or wherein said steroleosin is encoded by SEQ.ID.NO.:5.
6. A method according to claim 1 wherein said epidermal growth factor is human or porcine epidermal growth factor.
7. A chimeric nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in reading frame to (ii) a nucleic acid sequence encoding an epidermal growth factor.
8. A chimeric nucleic acid sequence according to claim 7 wherein said oil body protein is an oleosin, caleosin or steroleosin.
9. A chimeric nucleic acid sequence according to claim 8 wherein said oleosin is SEQ.ID.NO.: 1 or SEQ.ID.NO.: 2, wherein said caleosin is encoded by SEQ.ID.NO.:3 or SEQ.ID.NO.: 4 or wherein said steroleosin is encoded by SEQ.ID.NO.:5.
10. A chimeric nucleic acid sequence according to claim 7 having the nucleic acid sequence shown in SEQ.ID.NO.:10.
11. A chimeric nucleic acid sequence according to claim 7 wherein said epidermal growth factor is a human or porcine epidermal growth factor.
12. A chimeric nucleic acid sequence, capable of being expressed in association with an oil body of a plant cell, comprising:
(1) a first nucleic acid sequence capable of regulating the transcription in said plant cell;
(2) a second nucleic acid sequence, wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in reading frame to (ii) a nucleic acid sequence encoding an epidermal growth factor; and
(3) a third nucleic acid sequence encoding a termination region functional in said plant cell.
13. A chimeric nucleic acid sequence according to claim 12 wherein said oil body protein is an oleosin, caleosin or steroleosin.
14. A chimeric nucleic acid sequence according to claim 13 wherein said oleosin is SEQ.ID.NO.: 1 or SEQ.ID.NO.: 2, wherein said caleosin is encoded by SEQ.ID.NO.:3 or SEQ.ID.NO.: 4 or wherein said steroleosin is encoded by SEQ.ID.NO.:5.
15. A chimeric nucleic acid sequence according to claim 12 wherein said epidermal growth factor is a human or porcine epidermal growth factor.
16. A plant transformed with a chimeric nucleic acid sequence according to claim 12 .
17. A plant according to claim 16 wherein said plant is selected from the group consiting of rapeseed ( Brassica spp.), linseed/flax ( Linum usitatissimum ), safflower ( Carthamus tinctorius ), sunflower ( Helianthus annuus ), maize ( Zea mays ), soybean ( Glycine max ), mustard ( Brassica spp. and Sinapis alba ), crambe, ( Crambe abyssinica ), eruca ( Eruca sativa ), oil palm ( Elaeis guineeis ), cottonseed ( Gossypium spp.), groundnut ( Arachis hypogaea ), coconut ( Cocus nucifera ), castor bean ( Ricinus communis ), coriander ( Coriandrum sativum ), squash, ( Cucurbita maxima ), Brazil nut ( Bertholletia excelsa ) and jojoba ( Simmondsia chinensis ).
18. A plant seed containing a chimeric nucleic acid sequence according to claim 12 .
19. A plant seed according to claim 18 wherein said seed is obtained from a dicotelydenous plant.
20. A plant seed according to claim 18 wherein said epidermal growth factor is expressed in the embryogenic tissue of the seed.
21. A plant seed comprising a recombinantly expressed epidermal growth factor, wherein said epidermal growth factor is expressed as a fusion protein with an oil body protein.
22. A plant seed according to claim 21 wherein said epidermal growth to factor is a human or porcine epidermal growth factor.
23. A plant seed according to claim 21 wherein said oil body protein is an oleosin, caleosin or steroleosin.
24. A plant seed according to claim 23 wherein said oleosin is SEQ.ID.NO.: 1 or SEQ.ID.NO.: 2, wherein said caleosin is encoded by SEQ.ID.NO.:3 or SEQ.ID.NO.: 4 or wherein said steroleosin is encoded by SEQ.ID.NO.:5.
25. A plant seed according to claim 21 wherein said epidermal growth factor is expressed in the embryogenic tissue of said seed.
26. A plant seed according to claim 21 wherein said plant seed is obtained from a dicotelydenous plant.
27. A plant seed according to claim 21 wherein said seed is exalbuminous seed.
28. A plant seed according to claim 21 wherein said plant seed is obtained from the group of plants consisting rapeseed ( Brassica spp.), linseed/flax ( Linum usitatissimum ), safflower ( Carthamus tinctorius ), sunflower ( Helianthus annuus ), maize ( Zea mays ), soybean ( Glycine max ), mustard ( Brassica spp. and Sinapis alba ), crambe, ( Crambe abyssinica ), eruca ( Eruca sativa ), oil palm ( Elaeis guineeis ), cottonseed ( Gossypium spp.), groundnut ( Arachis hypogaea ), coconut ( Cocus nucifera ), castor bean ( Ricinus communis ), coriander ( Coriandrum sativum ), squash, ( Cucurbita maxima ), Brazil nut ( Bertholletia excelsa ) and jojoba ( Simmondsia chinensis ).