US2004023281A1PendingUtilityA1

Production of lysosomal enzymes in plants by transient expression

66
Priority: Feb 26, 1988Filed: Jun 23, 2003Published: Feb 5, 2004
Est. expiryFeb 26, 2008(expired)· nominal 20-yr term from priority
Inventors:Thomas H. TurpenMonto KumagaiGregory PogueRobert L. ErwinLaurence K. Grill
C07K 14/005C12Y 304/21069C12Y 114/18001C12N 9/14C12P 41/003C12N 9/84C12N 9/78C12N 9/6459C12N 9/0071C12N 15/86C12N 9/2402C12N 15/8242C07K 14/445C12N 9/0059C07K 2319/00C12N 9/16C12N 2770/32722C12N 15/8216C12N 15/8289C12N 15/8203C12N 15/8257C12N 9/20C12N 9/1074C12N 9/18C12Y 302/01031C12N 9/2465C12Y 302/01022C12Y 302/01045C07K 14/415C12N 2770/00022
66
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Claims

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.

Claims

exact text as granted — not AI-modified
We claim:  
     
         1 . A polypeptide comprising the amino acid sequence of rGAL-12, wherein the C-terminus of said polypeptide is the amino sequence depicted in SEQ ID NO: 29  
     
     
         2 . The polypeptide according to  claim 1 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-12.  
     
     
         3 . The polypeptide according to  claim 1 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-12.  
     
     
         4 . The polypeptide according to  claim 1 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         5 . The polypeptide according to  claim 1 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         6 . A polynucleotide encoding the polypeptide according to  claim 1 .  
     
     
         7 . The polypeptide according to  claim 1 , wherein said polypeptide consists of the amino acid sequence of rGAL-12.  
     
     
         8 . The polypeptide according to  claim 7 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-12.  
     
     
         9 . The polypeptide according to  claim 7 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-12.  
     
     
         10 . The polypeptide according to  claim 7 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         11 . The polypeptide according to  claim 7  wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         12 . A polynucleotide encoding the polypeptide according to  claim 7 .  
     
     
         13 . A polypeptide comprising the amino acid sequence of rGAL-12R, wherein the C-terminus of said polypeptide is the amino sequence depicted in SEQ ID NO: 30.  
     
     
         14 . The polypeptide according to  claim 13 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-12R.  
     
     
         15 . The polypeptide according to  claim 13 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-12R.  
     
     
         16 . The polypeptide according to  claim 13 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12R, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         17 . The polypeptide according to  claim 13 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12R, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         18 . A polynucleotide encoding the polypeptide according to  claim 13 .  
     
     
         19 . The polypeptide according to  claim 13 , wherein said polypeptide consists of the amino acid sequence of rGAL-12R.  
     
     
         20 . The polypeptide according to  claim 19 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-12R.  
     
     
         21 . The polypeptide according to  claim 19 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-12R.  
     
     
         22 . The polypeptide according to  claim 19 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12R, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         23 . The polypeptide according to  claim 19 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-12R, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         24 . A polynucleotide encoding the polypeptide according to  claim 9 .  
     
     
         25 . The polypeptide according to  claim 19 , wherein said polypeptide comprises the amino acid sequence of rGAL-25, wherein the C-terminus of said polypeptide is the amino sequence depicted in SEQ ID NO: 31.  
     
     
         26 . The polypeptide according to  claim 25 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-25.  
     
     
         27 . The polypeptide according to  claim 25 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-25.  
     
     
         28 . The polypeptide according to  claim 25 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         29 . The polypeptide according to  claim 25 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         30 . A polynucleotide encoding the polypeptide according to  claim 25 .  
     
     
         31 . The polypeptide according to  claim 25 , wherein said polypeptide consists of the amino acid sequence of rGAL-25.  
     
     
         32 . The polypeptide according to  claim 31 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-25.  
     
     
         33 . The polypeptide according to  claim 31 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-25.  
     
     
         34 . The polypeptide according to  claim 31 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         35 . The polypeptide according to  claim 31 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         36 . A polynucleotide encoding the polypeptide according to  claim 31 .  
     
     
         37 . The polypeptide according to  claim 19 , wherein said polypeptide comprises the amino acid sequence of rGAL-25R, wherein the C-terminus of said polypeptide is the amino sequence depicted in SEQ ID NO: 32.  
     
     
         38 . The polypeptide according to  claim 37 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-25R.  
     
     
         39 . The polypeptide according to  claim 37 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-25R.  
     
     
         40 . The polypeptide according to  claim 37 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25R, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         41 . The polypeptide according to  claim 37 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25R, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         42 . A polynucleotide encoding the polypeptide according to  claim 37 .  
     
     
         43 . The polypeptide according to  claim 37 , wherein said polypeptide consists of the amino acid sequence of rGAL-25R.  
     
     
         44 . The polypeptide according to  claim 43 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337 relative to rGAL-25R.  
     
     
         45 . The polypeptide according to  claim 43 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184 relative to rGAL-25R.  
     
     
         46 . The polypeptide according to  claim 43 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25R, wherein an alpha 1, 3 fucose is present on the proximal GlcNac.  
     
     
         47 . The polypeptide according to  claim 43 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184 relative to rGAL-25R, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         48 . A polynucleotide encoding the polypeptide according to  claim 43.

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