Compositions and methods for producing polypeptides with a modified glycosylation pattern in plant cells

Abstract

The A method of modifying a glycosylation pattern of a polypeptide-of-interest in a plant or plant cell is provided. The method comprising expressing in a plant or plant cell transformed to express at least one glycosidase in a subcellular compartment, a nucleic acid sequence encoding the polypeptide-of-interest, such that the at least one glycosidase and the polypeptide-of-interest are co-localized to the subcellular compartment of the plant or plant cell, thereby modifying the glycosylation pattern of the polypeptide-of-interest in the plant or plant cell.

What is claimed is: 
     
         1 . A method of producing a human polypeptide-of-interest, the method comprising expressing in a plant or plant cell:
 at least one glycosidase translationally fused to a signal peptide for localization in a subcellular compartment of a plant or plant cell;   a nucleic acid sequence encoding the human polypeptide-of-interest translationally fused to a signal peptide for localization in a subcellular compartment of a plant or plant cell,   such that said at least one glycosidase and said human polypeptide-of-interest are co-localized to said subcellular compartment of the plant or plant cell.   
     
     
         2 . The method of  claim 1 , further comprising an affinity moiety translationally fused to a cell wall binding peptide, wherein said affinity moiety is for binding said human polypeptide of interest; and wherein said affinity moiety is translationally fused to a nucleic acid sequence encoding a cell wall binding peptide. 
     
     
         3 . The method of  claim 2 , wherein said cell wall binding peptide is a cellulose binding domain (CBD). 
     
     
         4 . The method of  claim 1 , wherein said plant or plant cell is transformed to express said at least one glycosidase and wherein said expressing comprises introducing said nucleic acid sequence encoding the human polypeptide-of-interest into said transformed plant or plant cell. 
     
     
         5 . The method of  claim 1 , wherein said expressing comprises crossing:
 (i) a first transgenic plant transformed to express said at least one glycosidase; and   (ii) a second transgenic plant transformed to express the polypeptide of interest.   
     
     
         6 . The method of  claim 1 , wherein said expression comprises co-transforming said at least one glycosidase and said nucleic acid sequence encoding the human polypeptide-of-interest in said plant or plant cell. 
     
     
         7 . The method of  claim 1 , further comprising isolating the human polypeptide-of-interest from said plant or plant cell, thereby obtaining a non-fucosylated and/or non-xylosylated polypeptide. 
     
     
         8 . The method of  claim 1 , wherein said plant or plant cell has reduced level or activity of Beta-(l-2)-xylosyltransferase, Alpha-(1, 3)-fucosyltransferase and/or GDP-D-mannose 4,6-dehydratase as compared to a plant or plant cell of the same species expressing wild-type levels or exhibiting wild-type activity of said Beta-(l-2)-xylosyltransferase, Alpha-(1, 3)-fucosyltransferase and/or GDP-D-mannose 4,6-dehydratase. 
     
     
         9 . The method of  claim 1 , wherein said at least one glycosidase comprises a fucosidase and/or a xylosidase. 
     
     
         10 . The method of  claim 1 , wherein said signal peptide is a vacuolar signal peptide or an apoplast signal peptide. 
     
     
         11 . The method of  claim 1 , wherein said polypeptide-of interest is an antibody or an antibody fragment. 
     
     
         12 . The method of  claim 11 , wherein said antibody is bevacizumab or adalimumab. 
     
     
         13 . A nucleic acid construct system comprising one or more nucleic acid constructs, said one or more nucleic acid constructs comprising a nucleic acid sequence encoding a human polypeptide of interest; wherein said human polypeptide of interest is translationally fused to a vacuolar signal peptide or an apoplast signal peptide for localization thereof to a vacuole or an apoplast of a plant or plant cell. 
     
     
         14 . The nucleic acid construct system of  claim 13 , wherein said one or more nucleic acid constructs comprises a nucleic acid sequence encoding at least one glycosidase translationally fused to a signal peptide for localization to a subcellular compartment of a plant or plant cell and/or a nucleic acid sequence encoding an affinity moiety translationally fused to a cell wall binding peptide, wherein said affinity moiety is for binding said polypeptide of interest. 
     
     
         15 . The nucleic acid construct system  14 , wherein said cell wall binding peptide is a cellulose binding domain (CBD). 
     
     
         16 . The nucleic acid construct system of  claim 14 , wherein said polypeptide-of interest is an antibody or an antibody fragment. 
     
     
         17 . The nucleic acid construct system of  claim 16 , wherein said antibody is bevacizumab or adalimumab. 
     
     
         18 . The nucleic acid construct system of  claim 14 , wherein said at least one glycosidase comprises a fucosidase and/or a xylosidase. 
     
     
         19 . A transgenic plant or plant cell recombinantly expressing:
 (i) a human polypeptide of interest; and   (ii) at least one glycosidase   wherein each of said human polypeptide of interest and said at least one glycosidase is translationally fused to a signal peptide for localization to a vacuole or an apoplast of the plant or plant cell; such that a non-fucosylated and/or non-xylosylated human polypeptide is obtained.   
     
     
         20 . The transgenic plant of plant cell of  claim 19 , having reduced level or activity of Beta-(1-2)-xylosyltransferase, Alpha-(1, 3)-fucosyltransferase and/or GDP-D-mannose 4,6-dehydratase as compared to a plant or plant cell of the same species expressing wild-type levels or exhibiting wild-type activity of said Beta-(1-2)-xylosyltransferase, Alpha-(1, 3)-fucosyltransferase and/or GDP-D-mannose 4,6-dehydratase.