Glcnac phosphotransferase of the lysosomal targeting pathway
AssigneeGenzyme Glycobiology Research Institute, Inc.
InventorWilliam M. CANFIELD
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Abstract
The present invention provides nucleotide and amino sequences of the lysosomal targeting pathway enzyme GlcNAc-phosphotransferase, methods of producing and methods of purifying this enzyme.
Claims39
An isolated GlcNAc-phosphotransferase.
The GlcNAc-phosphotransferase wherein the α-subunit comprises the amino acid sequence in SEQ ID NO:1, the β-subunit comprises the amino acid sequence in SEQ ID NO:2, and the γ-subunit comprises the amino acid sequence in SEQ ID NO:3.
A method of producing a GlcNAc-phosphotransferase in a cell comprising
- transfecting into said cell a DNA construct comprising a targeting sequence homologous to a target site within or upstream of a endogenous GlcNAc-phosphotransferase gene contained in the cell, wherein said endogenous gene comprises the sequence in SEQ ID NO:4 or SEQ ID NO:5, an exogenous regulatory sequence, an exon, and an unpaired splice-donor site at the 3′ end of the exon, wherein said transfecting generates a homologously recombinant cell in which the splice-donor site is operatively linked to the second exon of the endogenous gene, and the exogenous regulatory sequence controls transcription of the construct driven, the endogenous gene, and any sequence lying between the construct-driven exon and the endogenous gene, to produce a RNA transcript that encodes the GlcNAc-phosphotransferase, so that the homologously recombinant cell produces the GlcNAc-phosphotransferase.
An isolated amino acid sequence comprising SEQ ID NO:1
A vector comprising the isolated nucleic acid of 23.
An isolated amino acid sequence comprising SEQ ID NO:2
A vector comprising the isolated nucleic acid of 29.
An isolated amino acid sequence comprising SEQ ID NO:3.
A vector comprising the isolated nucleic acid of 35.
Dependencies are inferred from the claim text.