Expression of human milk proteins in transgenic plants
The invention is directed to seed and seed extract compositions containing levels of a human milk protein between 3-40% or higher of the total protein weight of the soluble protein extractable from the seed. Also disclosed is a method of producing the seed with high levels of extractable human milk protein. The method includes transforming a monocotyledonous plant with a chimeric gene having a protein-coding sequence encoding a protein normally present in human milk under the control of a seed maturation-specific promoter. The method may further includes a leader DNA sequence encoding a monocot seed-specific transit sequence capable to target a linked milk protein to a storage body.
1. A method for making a cell culture medium comprising the steps of:
(a) obtaining a monocot plant stably transformed with a chimeric gene having (i) a seed maturation-specific promoter, (ii) operably linked to said promoter, a leader DNA sequence encoding a monocot seed-specific transit sequence capable of targeting a linked polypeptide to an endosperm-cell organelle, wherein the promoter operably linked to the leader sequence comprises either SEQ ID NO:13 or SEQ ID NO:14 and (iii) a protein-coding sequence encoding a protein normally present in human milk and having growth factor activity,
(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 the milk protein,
(e) purifying the milk protein from the aqueous solution, and
(f) adding the purified milk protein to cell culture medium.
2. The method of claim 1 , wherein the protein having growth factor activity is selected from the group consisting of human lactoferrin, EGF, IGF-1, albumin, casein and transferrin.
3. The method of claim 2 , wherein the protein having growth factor activity is human lactoferrin.
4. The method of claim 1 , wherein the protein-coding sequence is the codon- optimized sequence identified by SEQ ID NO: 3.
5. The method of claim 1 , wherein the transformed monocot plant is a rice plant, and the promoter operably linked to the leader sequence comprises SEQ ID NO: 13.
6. The method of claim 1 , wherein the transformed monocot plant is a rice plant, and the promoter operably linked to the leader sequence comprises SEQ ID NO:14.
7. The method of claim 1 , wherein the transformed monocot plant further comprises a nucleic acid that encodes at least one transcription factor selected from the group consisting of Reb, O2 and PBF.
8. The method of claim 7 , wherein the transcription factor is O2 and/or PBF.
9. The method of claim 1 , wherein the human milk protein coding sequence is selected from the group of codon-optimized sequences identified by SEQ ID NOS: 1, 3, and 7-12.