Expression of lysosomal hydrolase in cells expressing pro-N-acetylglucosamine-1-phosphodiester alpha-N-acetyl glucosimanidase
AssigneeNovazyme Pharmaceuticals, Inc.
InventorsWilliam Canfield, Stuart Kornfeld
Life of this patent
Estimating the term…
Abstract
The present invention provides methods of producing a pro-N-acetylglucosamine-1-phosphodiester α N-acetyl glucosimanidase (phosphodiester α-GlcNAcase), in mammalian cells deficient in the furin proteolytic enzyme and methods of making lysosomal hydrolases having an N-acetylglucosamine-1-phosphate.
Claims44
A method of producing a lysosomal hydrolase having an oligosaccharide modified with N-acteylglucosamine-1-phosphate comprising
- a. contacting a mammalian cell culture expressing a lysosomal hydrolase with a mutagenic agent;
- b. culturing said mammalian cell culture in the presence of Pseudomonas exotoxin A in an amount sufficient to select for cells resistant to the Pseudomonas exotoxin A;
- c. selecting said cells resistant to Pseudomonas exotoxin A;
- and
- d. isolating said lysosomal hydrolase having an N-acteylglucosamine-1-phosphate from said resistant cells.
A method of producing a lysosomal hydrolase having an oligosaccharide modified with N-acteylglucosamine-1-phosphate comprising
- a. introducing a polynucleotide sequence encoding the lysosomal hydrolase in a furin deficient mammalian cell;
- b. culturing said furin deficient mammalian cell containing the polynucleotide sequence encoding the lysosomal hydrolase for a time and under conditions suitable for expression of the lysosomal hydrolase;
- and
- c. collecting the lysosomal hydrolase expressed.
A method of producing a lysosomal hydrolase having an oligosaccharide N-acteylglucosamine-1-phosphate comprising
- a. a step for expressing a lysosomal hydrolase in a furin deficient mammalian cell;
- and
- b. a step for collecting the lysosomal hydrolase expressed.
A method of producing a phosphodiester α-GlcNAcase comprising
- a. contacting a mammalian cell culture expressing said phosphodiester α-GlcNAcase with a mutagenic agent;
- b. culturing said mammalian cell culture in the presence of Pseudomonas exotoxin A in an amount sufficient to select for cells resistant to the Pseudomonas exotoxin A;
- c. selecting said cells resistant to Pseudomonas exotoxin A;
- and
- d. isolating said phosphodiester α-GlcNAcase having a pro-peptide from said resistant cells.
A method of producing a phosphodiester β-GlcNAcase comprising
- a. culturing a furin deficient mammalian cell expressing said phosphodiester α-GlcNAcase for a time and under conditions suitable for expression of the phosphodiester α-GlcNAcase;
- and
- b. collecting the lysosomal hydrolase expressed.
A method of producing a phosphodiester α-GlcNAcase comprising
- a. a step for expressing said phosphodiester α-GlcNAcase in a furin deficient mammalian cell;
- and
- b. a step for collecting the phosphodiester α-GlcNAcase expressed.
A method of producing a lysosomal hydrolase having an oligosaccharide modified N-acteylglucosamine-1-phosphate comprising
- a. culturing a mammalian cell in the presence of Pseudomonas exotoxin A in an amount sufficient to select for cells resistant to the Pseudomonas exotoxin A;
- b. selecting said cells resistant to Pseudomonas exotoxin A;
- and
- c. isolating said lysosomal hydrolase having an N-acteylglucosamine-1-phosphate from said resistant cells.
A method of producing a phosphodiester α-GlcNAcase comprising
- a. culturing said mammalian cell culture in the presence of Pseudomonas exotoxin A in an amount sufficient to select for cells resistant to the Pseudomonas exotoxin A;
- b. selecting said cells resistant to Pseudomonas exotoxin A;
- and
- c. isolating said phosphodiester α-GlcNAcase having a pro-peptide from said resistant cells.
Dependencies are inferred from the claim text.