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US 6,670,165 B2Granted
Methods for producing highly phosphorylated lysosomal hydrolases
AssigneeGenzyme Glycobiology Research Institute, Inc.
InventorWilliam M. Canfield
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Abstract
The present invention provides highly phosphorylated lysosomal hydrolases, methods of modifying lysosomal hydrolases with the lysosomal targeting pathway enzymes GlcNAc-phosphotransferase and/or phosphodiester α-GlcNAcase.
Claims42
A method of producing a modified lysosomal hydrolase comprising
- culturing transformed cells comprising a recombinant polynucleotide encoding a lysosomal hydrolase in the presence of an α1,2-mannosidase inhibitor;
- recovering the lysosomal hydrolase from the cultured transformed cells;
- and
- contacting the recovered lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphotransferase to produce a modified lysosomal hydrolase, wherein said N-acetylglucosamine-phosphotransferase comprises SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
1 more element
A method of modifying a lysosomal hydrolase comprising
- culturing transformed cells comprising a recombinant polynucleotide encoding a lysosomal hydrolase in the presence of an α1,2-mannosidase inhibitor;
- recovering the lysosomal hydrolase from the cultured transformed cells;
- and
- contacting said recovered lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphotransferase to produce a modified lysosomal hydrolase, wherein said N-acetylglucosamine-phosphotransferase comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1, an amino acid sequence that is at least 70% identical to SEQ ID NO:2, and an amino acid sequence that is at least 70% identical to SEQ ID NO:3, and said N-acetylglucosamine-phosphotransferase catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-N-Acetylglucosamine to a mannose on the lysosomal hydrolase.
1 more element
Dependencies are inferred from the claim text.