MILK PROTEIN PRODUCTION IN TRANSGENIC PLANTS
The disclosure describes a transgenic dicot or monocot plant having bovine milk protein(s) and methods of producing the transgenic dicot or monocot plant containing bovine milk protein(s). These transgenic dicot or monocot plants can express and produce bovine milk protein(s). The methods involve introducing a recombinant DNA construct expressing a bovine milk protein into a dicot or monocot plant, obtaining the dicot or monocot plant containing the bovine milk protein(s) from a recombinant DNA construct, cultivating and harvesting the transgenic dicot or monocot plant, and extracting and purifying the bovine milk protein(s) from transgenic dicot or monocotyledonous plants.
1 . A stably transformed dicotyledonous plant comprising in its genome a recombinant DNA construct, said construct comprising:
(i) a promoter,
(ii) a nucleic acid sequence encoding a bovine κ-casein milk protein having at least 90% sequence identity to SEQ ID NO:5, which is operably linked to said promoter, and
(iii) a termination sequence;
wherein the bovine milk protein is stably expressed in the plant and/or a part thereof in an amount of 1% or higher per the total protein weight of the soluble protein extractable from the plant or part thereof.
2 . The stably transformed dicotyledonous plant of claim 1 , wherein the plant is selected from the group consisting of soybean, lima bean, Arabidopsis , and tobacco.
3 . The stably transformed dicotyledonous plant of claim 1 , wherein the promoter is selected from a Cauliflower mosaic virus (CaMV) 35S promoter, a plant constitutive promoter, and a plant tissue-specific promoter.
4 . The stably transformed dicotyledonous plant of claim 3 , wherein the plant constitutive promoter comprises a nucleic acid having at least 90% sequence identity to SEQ ID No: 49.
5 . The stably transformed dicotyledonous plant of claim 3 , wherein the plant tissue-specific promoter comprises a nucleic acid having at least 90% sequence identity to SEQ ID No: 32.
6 . The stably transformed dicotyledonous plant of claim 1 , wherein the nucleic acid sequence encoding the bovine κ-casein milk protein is codon-optimized.
7 .- 19 . (canceled)
20 . The stably transformed dicotyledonous plant of claim 1 , wherein the termination sequence is a NOS terminator.
21 .- 22 . (canceled)
23 . The stably transformed dicotyledonous plant of claim 1 , wherein the bovine milk protein further comprises peptides produced by proteolysis of κ-casein.
24 . A method of producing the stably transformed dicotyledonous plant of claim 1 , said method comprising the steps of:
(a) introducing at least one expression cassette capable of expressing a bovine κ-casein milk protein into a plant, a part thereof, or a cell thereof,
(b) obtaining the stably transformed dicotyledonous plant, the part thereof, or the cell thereof, which stably expresses the bovine κ-casein milk protein,
(c) cultivating the stably transformed dicotyledonous plant, the part thereof, or the cell thereof,
(d) harvesting the stably transformed dicotyledonous plant, the part thereof, or the cell thereof.
25 . A method of producing a bovine κ-casein milk protein from the stably transformed dicotyledonous plant of claim 1 , said method comprising the steps of:
(a) extracting the bovine κ-casein milk protein from the stably transformed dicotyledonous plant, the part thereof, or the cell thereof, and
(b) purifying the bovine κ-casein milk protein from the stably transformed dicotyledonous plant, the part thereof, or the cell thereof.
26 . A stably transformed dicotyledonous plant comprising in its genome a recombinant DNA construct, said construct comprising:
(i) a promoter,
(ii) a nucleic acid sequence encoding a bovine milk protein, which is operably linked to said promoter, and
(iii) a termination sequence;
wherein the bovine milk protein is stably expressed in the plant and/or a part thereof in an amount of 1% or higher per the total protein weight of the soluble protein extractable from the plant or part thereof; and
wherein the bovine milk protein is selected from the group consisting of α-S1 casein, α-S2 casein, β-casein, α-lactalbumin, β-lactoglobulin, and lysozyme.
27 . The stably transformed dicotyledonous plant of claim 26 , wherein the plant is selected from the group consisting of soybean, lima bean, Arabidopsis , and tobacco.
28 . The stably transformed dicotyledonous plant of claim 26 , wherein the promoter is selected from a Cauliflower mosaic virus (CaMV) 35S promoter, a plant constitutive promoter, and a plant tissue-specific promoter.
29 . The stably transformed dicotyledonous plant of claim 28 , wherein the plant constitutive promoter comprises a nucleic acid having at least 90% sequence identity to SEQ ID No: 49.
30 . The stably transformed dicotyledonous plant of claim 28 , wherein the plant tissue-specific promoter comprises a nucleic acid having at least 90% sequence identity to SEQ ID No: 32.
31 . The stably transformed dicotyledonous plant of claim 26 , wherein the nucleic acid sequence encoding the bovine milk protein is codon-optimized.
32 . The stably transformed dicotyledonous plant of claim 26 , wherein the bovine milk protein has a sequence with at least 90% sequence identity to any one of SEQ ID No: 7, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 22, SEQ ID No: 23, or SEQ ID No: 24.
33 . The stably transformed dicotyledonous plant of claim 26 , wherein the termination sequence is a NOS terminator.
34 . The stably transformed dicotyledonous plant of claim 26 , wherein the bovine milk protein further comprises peptides produced by proteolysis of α-S1 casein, α-S2 casein, f3-casein, α-lactalbumin, β-lactoglobulin, or lysozyme.
35 . A method of producing the stably transformed dicotyledonous plant of claim 26 , said method comprising the steps of:
(a) introducing at least one expression cassette capable of expressing a bovine milk protein into a plant, a part thereof, or a cell thereof,
(b) obtaining the stably transformed dicotyledonous plant, the part thereof, or the cell thereof, which stably expresses the bovine milk protein,
(c) cultivating the stably transformed dicotyledonous plant, the part thereof, or the cell thereof,
(d) harvesting the stably transformed dicotyledonous plant, the part thereof, or the cell thereof.
36 . A method of producing a bovine milk protein from the stably transformed dicotyledonous plant of claim 26 , the method comprising the steps of:
(a) extracting the bovine milk protein from the stably transformed dicotyledonous plant, the part thereof, or the cell thereof, and
(b) purifying the bovine milk protein from the stably transformed dicotyledonous plant, the part thereof, or the cell thereof.