Gene expression and production of TGF-beta proteins including bioactive mullerian inhibiting substance from plants

Abstract

This invention describes a novel method of producing bioactive recombinant proteins from plants. General methods of designing and engineering plants for expression and production of such proteins are also disclosed. Methods for the expression of Transforming Growth Factor-β (TGF-β) proteins, such as Müllerian Inhibiting Substance (MIS), in plants, and methods of producing recombinant proteins from plants are specifically disclosed. Furthermore, the present invention provides methodology for the direct expression and production of a bioactive C-terminal fragment of a TGF-β protein, such as C-terminal MIS. The new method is more cost-effective than other large-scale expression systems, by eliminating the need for costly cell culture and fermentation manufacturing facilities.

We claim:  
     
         1 . A method for producing a recombinant TGF-β protein in a plant host system, comprising 
 transforming said plant host system with a chimeric nucleic acid sequence that encodes a TGF-β protein;  
 cultivating a transformed host system under conditions to express the TGF-β protein; and  
 recovering an expressed TGF-β protein from said plant host system.  
 
     
     
         2 . The method of  claim 1 , wherein said TGF-β protein is selected from a group consisting of Activins, MIS, BMP and MNSF proteins.  
     
     
         3 . The method of  claim 1 , wherein said TGF-β protein is a human MIS protein.  
     
     
         4 . The method of  claim 1 , wherein said expressed TGF-β protein is a full length TGF-β protein, a TGF-β peptide fragment, or a genetic or chemical variant thereof.  
     
     
         5 . The method of  claim 4 , wherein said peptide fragment comprises a bioactive C-terminal fragment.  
     
     
         6 . The method of  claim 5 , wherein said bioactive C-terminal fragment is free from a plant-specific glycan.  
     
     
         7 . The method of  claim 1 , wherein said expressed TGF-β protein contains a N-linked glycan comprising a plant-specific glycan or a glycan composition.  
     
     
         8 . The method of  claim 7 , further comprising modifying said N-linked glycan in vivo or in vitro to reduce or eliminate a plant-specific glycan or to produce a more desirable glycovariant.  
     
     
         9 . The method of  claim 8 , wherein a TGF-β protein recovered from a plant host system is treated by a chemical or an enzyme.  
     
     
         10 . The method of  claim 9 , wherein said enzyme comprises a glycosidase or a glycosyltransferase.  
     
     
         11 . The method of  claim 10 , wherein said glycosidase is selected from a group consisting of a xylosidase and 1,3-fucosidase and said glycosyltransferase is selected from a group consisting of a galatosyltransferase or a sialyltransferase.  
     
     
         12 . The method of  claim 1 , wherein said chimeric nucleic acid sequence comprises a first nucleic acid sequence capable for regulating transcription in said plant host system, a second nucleic acid sequence encoding a signal sequence and a third nucleic acid sequence encoding the TGF-β protein.  
     
     
         13 . The method of  claim 11 , wherein said third nucleic acid sequence comprises a gene for encoding a full length TGF-β protein or a bioactive TGF-β protein C-terminal fragment.  
     
     
         14 . The method of  claim 13 , said TGF-β protein is a human MIS protein.  
     
     
         15 . The method of  claim 12 , wherein said third nucleic acid sequence comprises a gene for encoding a full-length protein and a cleavage site.  
     
     
         16 . The method of  claim 15 , wherein said cleavage site is cleavable by enzymatic or chemical means.  
     
     
         17 . The method of  claim 12 , wherein said first nucleic acid is a plant-active promoter.  
     
     
         18 . The method of  claim 17 , wherein said plant-active promoter is derived from plant gene sequences, plant viral sequences, non-plant gene sequences or synthetic origin.  
     
     
         19 . The method of  claim 17 , wherein said plant-active promoter is selected from a group consisting of a constitutive promoter, a pre-harvest inducible promoter, a post-harvest inducible promoter, a developmentally regulated promoter, or a tissues-specific promoter.  
     
     
         20 . The method of  claim 17 , wherein said plant-active promoter is the MeGA™ promoter.  
     
     
         21 . The method of  claim 12 , wherein said second nucleic acid sequence targets said TGF-β protein to a sub-cellular location within the plant host system.  
     
     
         22 . The method of  claim 21 , wherein said sub-cellular location comprises the cytosol, plastid, endoplasmic reticulum or the apoplast.  
     
     
         23 . The method of  claim 12 , wherein said second nucleic acid sequence comprises a gene for encoding a plant-derived signal peptide.  
     
     
         24 . The method of  claim 23 , wherein said plant-derived signal peptide is the patatin signal peptide.  
     
     
         25 . An expression cassette comprising a first nucleic acid sequence capable for regulating transcription in said plant host system, a second nucleic acid sequence encoding a signal sequence and a third nucleic acid sequence encoding the TGF-β protein.  
     
     
         26 . The expression cassette of  claim 25 , wherein said third nucleic acid sequence comprises a gene for encoding a full length TGF-β protein or a bioactive TGF-β protein C-terminal fragment.  
     
     
         27 . The expression cassette of  claim 26 , said TGF-β protein is human MIS protein.  
     
     
         28 . The expression cassette method of  claim 25 , wherein said third nucleic acid sequence comprises a gene for encoding a full-length protein and a cleavage site.  
     
     
         29 . The expression cassette method of  claim 28 , wherein said cleavage site is cleavable by enzymatic or chemical means.  
     
     
         30 . The expression cassette method of  claim 25 , wherein said first nucleic acid is a plant-active promoter.  
     
     
         31 . The expression cassette of  claim 30 , wherein said plant-active promoter is derived from plant gene sequences, plant viral sequences, non-plant gene sequences or synthetic origin.  
     
     
         32 . The expression cassette of  claim 30 , wherein said plant-active promoter is selected from a group consisting of a constitutive promoter, a pre-harvest inducible promoter, a post-harvest inducible promoter, a developmentally regulated promoter, or a tissues-specific promoter.  
     
     
         33 . The expression cassette method of  claim 30 , wherein said plant-active promoter is the MeGA™ promoter.  
     
     
         34 . The expression cassette method of  claim 25 , wherein said second nucleic acid sequence targets said TGF-β protein to a sub-cellular location within the plant host system.  
     
     
         35 . The expression cassette method of  claim 34 , wherein said sub-cellular location comprises the cytosol, plastid, endoplasmic reticulum or the apoplast.  
     
     
         36 . The expression cassette method of  claim 25 , wherein said second nucleic acid sequence comprises a gene for encoding a plant-derived signal peptide.  
     
     
         37 . The expression cassette method of  claim 36 , wherein said plant-derived signal peptide is the patatin signal peptide.  
     
     
         38 . A plant host system transformed with the expression cassette of  claim 25 .  
     
     
         39 . A plant host system transformed with the expression cassette of  claim 26 .  
     
     
         40 . A plant host system transformed with the expression cassette of  claim 27.