Production of lysosomal enzymes in plants by transient expression

Abstract

The invention relates to α-galactosidase truncated at the carboxy terminus and the production of enzymatically active recombinant human and animal lysosomal enzymes involving construction and expression of recombinant expression constructs comprising coding sequences of human or animal lysosomal enzymes in a plant expression system. The plant expression system provides for post-translational modification and processing to produce a recombinant gene product exhibiting enzymatic activity. The invention is demonstrated by working examples in which transgenic tobacco plants express recombinant expression constructs comprising human glucocerebrosidase nucleotide sequences. The invention is also demonstrated by working examples in which transfected tobacco plants express recombinant viral expression constructs comprising human α galactosidase nucleotide sequences. The recombinant lysosomal enzymes produced in accordance with the invention may be used for a variety of purposes, including but not limited to enzyme replacement therapy for the therapeutic treatment of human and animal lysosomal storage diseases.

1 - 21 . (canceled)  
     
     
         22 . An isolated polypeptide comprising: 
 (a) an amino acid sequence of an α-galactosidase having a deletion at the carboxy end of α-galactosidase, said deletion being one to twenty-five amino acids.    (b) the amino acid depicted in SEQ ID NO: 27 at the carboxy end of said amino acid sequence of α-galactosidase.    
     
     
         23 . The isolated polypeptide according to  claim 22 , wherein said deletion is one to twelve amino acids.  
     
     
         24 . The isolated polypeptide according to  claim 22 , wherein said polypeptide is glycosylated at least at one amino acid position.  
     
     
         25 . The isolated polypeptide according to  claim 22 , wherein said polypeptide has a plant glycosylation pattern  
     
     
         26 . The isolated polypeptide according to  claim 22 , wherein said deletion is four to twelve amino acids.  
     
     
         27 . A plant cell or a plant expressing an isolated polypeptide comprising an amino acid sequence of an α-galactosidase as depicted in SEQ ID NO: 12.  
     
     
         28 . An isolated polynucleotide comprising a nucleotide sequence encoding an isolated polypeptide comprising an amino acid sequence of an α-galactosidase as depicted in SEQ ID NO: 12.  
     
     
         29 . A plant cell or a plant expressing the polypeptide according to  claim 22 .  
     
     
         30 . An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide according to  claim 22.