Recombinant fusion proteins for producing milk proteins in plants

Abstract

Provided herein are compositions and methods for producing milk proteins in plants, which allow for safe, sustainable and humane production of milk proteins for commercial use, such as use in food compositions. The disclosure provides recombinant fusion proteins comprising a milk protein, or fragment thereof and a structured mammalian, avian, plant, or fungal protein, or fragment thereof. The disclosure also provides methods for producing the recombinant fusions proteins, and food compositions comprising the same.

1 . A transformed plant comprising in its genome: a recombinant DNA construct encoding a fusion protein, the fusion protein comprising:
 (i) an unstructured milk protein, and   (ii) a structured animal protein.   
     
     
         2 . The transformed plant of  claim 1 , wherein the structured animal protein is β-lactoglobulin, α-lactalbumin, albumin, lysozyme, lactoferrin, lactoperoxidase, hemoglobin, collagen, or an immunoglobulin. 
     
     
         3 . The transformed plant of  claim 1 , wherein the fusion protein comprises, from N-terminus to C-terminus, the unstructured milk protein and the structured animal protein. 
     
     
         4 . The transformed plant of  claim 1 , wherein the fusion protein comprises, from N-terminus to C-terminus, the structured animal protein and the unstructured milk protein. 
     
     
         5 . The transformed plant of  claim 1 , wherein the unstructured milk protein is selected from the group consisting of α-S1 casein, α-S2 casein, β-casein, and κ-casein. 
     
     
         6 . The transformed plant of  claim 1 , wherein the unstructured milk protein is κ-casein and comprises the sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. 
     
     
         7 . The transformed plant of  claim 1 , wherein the unstructured milk protein is para-κ-casein and comprises the sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto. 
     
     
         8 . The transformed plant of  claim 1 , wherein the unstructured milk protein is β-casein and comprises the sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto. 
     
     
         9 . The transformed plant of  claim 1 , wherein the unstructured milk protein is α-S1 casein and comprises the sequence SEQ ID NO: 8, or a sequence at least 90% identical thereto. 
     
     
         10 . The transformed plant of  claim 1 , wherein the unstructured milk protein is α-S2 casein and comprises the sequence SEQ ID NO: 84, or a sequence at least 90% identical thereto. 
     
     
         11 . The transformed plant of  claim 1 , wherein the fusion protein is expressed in the plant in an amount of 1% or higher per total protein weight of soluble protein extractable from the plant. 
     
     
         12 . The transformed plant of  claim 1 , wherein the plant is a dicot. 
     
     
         13 . The transformed plant of  claim 12 , wherein the dicot is  Arabidopsis , tobacco, tomato, potato, sweet potato, cassava, alfalfa, lima bean, pea, chick pea, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, Quinoa, buckwheat, mung bean, cow pea, lentil, lupin, peanut, fava bean, french beans, mustard, or cactus. 
     
     
         14 . The transformed plant of  claim 1 , wherein the plant is soybean. 
     
     
         15 . The transformed plant of  claim 1 , wherein the recombinant DNA construct comprises codon-optimized nucleic acids for expression in the plant. 
     
     
         16 . The transformed plant of  claim 1 , wherein the fusion protein has a molecular weight of 30 kDa to 50 kDa. 
     
     
         17 . The transformed plant of  claim 1 , wherein the fusion protein is expressed at a level that is at least 2-fold higher than a control plant expressing the unstructured milk protein without the structured animal protein. 
     
     
         18 . A method of producing a fusion protein comprising extracting the fusion protein from the transformed plant of  claim 1 . 
     
     
         19 . The method of  claim 18 , wherein the extracted fusion protein is comprised within a food composition. 
     
     
         20 . The method of  claim 19 , wherein the food composition is cheese.