Human Blood Proteins Expressed in Monocot Seeds

Abstract

The invention is directed to blood proteins produced in monocot seeds and isolated therefrom for use in therapeutic compositions, and to methods of making these isolated blood proteins and to therapeutic compositions comprising them.

1 . A method of producing a recombinant human blood protein in monocot plant seeds, comprising the steps of:
 (a) transforming a monocot plant cell with a chimeric gene comprising
 (i) a promoter from the gene of a maturation-specific monocot plant storage protein, 
 (ii) a first DNA sequence, operably linked to said promoter, encoding a monocot plant seed-specific signal sequence capable of targeting a polypeptide linked thereto to a monocot plant seed endosperm cell, and 
 (iii) a second DNA sequence, linked in translation frame with the first DNA sequence, encoding a human blood protein, wherein the first DNA sequence and the second DNA sequence together encode a fusion protein comprising an N-terminal signal sequence and the human blood protein; 
   (b) growing a monocot plant from the transformed monocot plant cell for a time sufficient to produce seeds containing the human blood protein; and   (c) harvesting the seeds from the plant,   
       wherein the human blood protein constitutes at least about 3.0% of the total soluble protein in the harvested seeds. 
     
     
         2 . The method of  claim 1 , wherein the human blood protein constitutes at least about 5.0% of the total soluble protein in the harvested seeds. 
     
     
         3 . The method of  claim 1 , wherein the human blood protein constitutes at least about 10.0% of the total soluble protein in the harvested seeds. 
     
     
         4 . The method of  claim 1 , further comprising purifying the human blood protein from the harvested seeds. 
     
     
         5 . The method of  claim 1 , wherein the human blood protein is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antithrombin III, an antibody, and a blood coagulation factor. 
     
     
         6 . The method of  claim 1 , wherein the human blood protein produced in the method comprises one or more plant glycosyl groups. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . A monocot plant seed product selected from the group consisting of whole seed, flour, extract and malt, prepared from the harvested seeds obtained by the method of  claim 1 , wherein the human blood protein constitutes at least about 3.0% of the total soluble protein in the seed product. 
     
     
         10 . The seed product of  claim 9 , wherein the human blood protein constitutes at least about 5.0% of the total soluble protein in the seed product. 
     
     
         11 . The seed product of  claim 9 , wherein the human blood protein constitutes at least about 10.0% of the total soluble protein in the seed product. 
     
     
         12 . The seed product of  claim 9 , wherein the human blood protein is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antithrombin III, an antibody, and a blood coagulation factor. 
     
     
         13 - 18 . (canceled) 
     
     
         19 . A method of producing a recombinant antibody in monocot plant seeds, comprising the steps of:
 (a) transforming a monocot plant cell with a chimeric gene comprising
 (i) a promoter from the gene of a maturation-specific monocot plant storage protein, 
 (ii) a first DNA sequence, operably linked to said promoter, encoding a monocot plant seed-specific signal sequence capable of targeting a polypeptide linked thereto to a monocot plant seed endosperm cell, and 
 (iii) a second DNA sequence, linked in translation frame with the first DNA sequence, encoding an antibody, wherein the first DNA sequence and the second DNA sequence together encode a fusion protein comprising an N-terminal signal sequence and the antibody; 
   (b) growing a monocot plant from the transformed monocot plant cell for a time sufficient to produce seeds containing the antibody; and   (c) harvesting the seeds from the plant,   
       wherein the antibody constitutes at least about 3.0% of the total soluble protein in the harvested seeds. 
     
     
         20 . The method of  claim 19 , wherein the antibody is selected from the group consisting of IgG, IgA, and IgM. 
     
     
         21 . The method of  claim 19 , wherein the antibody constitutes at least about 5.0% of the total soluble protein in the harvested seeds. 
     
     
         22 . The method of  claim 19 , wherein the antibody constitutes at least about 10.0% of the total soluble protein in the harvested seeds. 
     
     
         23 . The method of  claim 19 , further comprising purifying the antibody from the harvested seeds. 
     
     
         24 . A method of producing a recombinant human blood protein in monocot plant seeds, comprising the steps of:
 (a) transforming a monocot plant cell with a first expression construct and a second expression construct, each comprising a chimeric gene which is different for each construct, each chimeric gene comprising
 (i) a promoter from the gene of a maturation-specific monocot plant storage protein, 
 (ii) a first DNA sequence, operably linked to said promoter, encoding a monocot plant seed-specific signal sequence capable of targeting a polypeptide linked thereto to a monocot plant seed endosperm cell, and 
 (iii) a second DNA sequence, linked in translation frame with the first DNA sequence, encoding a portion of a human blood protein, wherein the first DNA sequence and the second DNA sequence together encode a fusion protein comprising an N-terminal signal sequence and the portion of the human blood protein, 
   
       wherein the portions of the human blood protein are assembled to form the human blood protein;
 (b) growing a monocot plant from the transformed monocot plant cell for a time sufficient to produce seeds containing the human blood protein; and 
 (c) harvesting the seeds from the plant, 
 
       wherein the human blood protein constitutes at least about 3.0% of the total soluble protein in the harvested seeds. 
     
     
         25 . The method of  claim 24 , wherein the human blood protein is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antithrombin III, an antibody, and a blood coagulation factor. 
     
     
         26 . A method of producing a recombinant antibody in monocot plant seeds, comprising the steps of:
 (a) transforming a monocot plant cell with a first expression construct and a second expression construct, each comprising a chimeric gene which is different for each construct, each chimeric gene comprising
 (i) a promoter from the gene of a maturation-specific monocot plant storage protein, 
 (ii) a first DNA sequence, operably linked to said promoter, encoding a monocot plant seed-specific signal sequence capable of targeting a polypeptide linked thereto to a monocot plant seed endosperm cell, and 
 (iii) a second DNA sequence, linked in translation frame with the first DNA sequence, encoding a portion of an antibody, wherein the first DNA sequence and the second DNA sequence together encode a fusion protein comprising an N-terminal signal sequence and the portion of the antibody, 
   
       wherein the portions of the antibody are assembled to form the antibody;
 (b) growing a monocot plant from the transformed monocot plant cell for a time sufficient to produce seeds containing the antibody; and 
 (c) harvesting the seeds from the plant, 
 
       wherein the antibody constitutes at least about 3.0% of the total soluble protein in the harvested seeds. 
     
     
         27 . The method of  claim 26 , wherein the antibody is selected from the group consisting of IgG, IgA, and IgM. 
     
     
         28 . The method of  claim 1 , wherein the promoter is selected from the group consisting of a rice glutelin 1 (Gt1) promoter, a rice glutelin 3 (Gt3) promoter, a rice glutelin B1 (GluB-1) promoter, a rice glutelin B2 (GluB-2) promoter, a rice globulin (Glb) promoter, a rice prolamin (Rp-6) promoter, and a wheat glutenin (Bx7) promoter.