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Last Updated: November 29, 2021

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Claims for Patent: 5,491,075

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Summary for Patent: 5,491,075
Title: Cloning and expression of biologically active .alpha.-N-acetylgalactosaminidase
Abstract:The present invention involves the production of human .alpha.-GalNAc by cloning and expressing the .alpha.-GalNAc coding sequence in eukaryotic host cell expressions systems. The eukaryotic expression systems, and in particular the mammalian host cell expression systems described herein provide for the appropriate co-translational and post-translation modifications required or proper processing, e.g., glycosylation, phosphorylation, etc. and sorting of the expression product so that an active enzyme is produced. The .alpha.-GalNAc produced in accordance with the invention may be used in the treatment of Schindler disease or for the hydrolysis of .alpha.-N-acetylgalactosaminyl moieties in various glycoconjugates.
Inventor(s): Desnick; Robert J. (New York, NY), Bishop; David F. (New York, NY), Ioannou; Yiannis A. (New York, NY), Wang; Anne M. (New York, NY)
Assignee: The Mount Sinai School of Medicine of the City University of New York (New York, NY)
Application Number:08/261,578
Patent Claims:1. A method for producing human .alpha.-N-acetylgalactosaminidase, comprising:

a. culturing a mammalian cell containing a nucleotide sequence encoding a cleavage site sandwiched between an .alpha.-N-acetylgalactosaminidase coding sequence and a protein A domain E coding sequence arranged in the same translational reading frame controlled by a second nucleotide sequence that regulates gene expression so that a fusion protein is expressed by the cell;

b. recovering the fusion protein from the culture;

c. treating the fusion protein with a substance that cleaves the cleavage site so that the .alpha.-N-acetylgalactosaminidase is separated from the binding protein; and

d. recovering the separated .alpha.-N-acetylgalactosaminidase.

2. The method according to claim 1 in which the fusion protein is recovered from the culture by reaction with an immunoglobulin binding partner for the protein A domain E protein.

3. The method according to claim 2 in which the immunoglobulin binding partner is immobilized.

4. The method according to claim 1 in which the recombinant vector is pAGB-3 as deposited with the Agricultural Research Culture Collection having accession number B-18724.

5. A method for producing human .alpha.-N-acetylgalactosaminidase, comprising:

a. culturing a mammalian cell containing a nucleotide sequence encoding a cleavage site sandwiched between an .alpha.-N-acetylgalactosaminidase coding sequence and a protein antigen coding sequence arranged in the same translational reading frame, controlled by a second nucleotide sequence that regulates gene expression so that a fusion protein is expressed by the cell;

b. recovering the fusion protein from the culture by reaction with an immunoglobulin directed against the protein antigen;

c. treating the fusion protein with a substance that cleaves the cleavage site so that the .alpha.-N-acetylgalactosaminidase is separated from the protein antigen; and

d. recovering the separated .alpha.-N-acetylgalactosaminidase.

6. The method according to claim 5 in which the immunoglobulin is immobilized.

7. The method according to claim 1 or 5 in which the substance that cleaves the cleavage site is an enzyme and the cleavage site is a substrate specific for the enzyme.

8. The method according to claim 7 in which the enzyme is a collagenase.

9. The method according to claim 1 or 5 in which the .alpha.-N-acetylgalactosaminidase coding sequence comprises the sequence (SEQ ID NO: 1) depicted in FIG. 2 from nucleotide number 1 to 1236.

10. The method according to claim 1 or 5 in which the .alpha.-N-acetylgalactosaminidase coding sequence comprises the sequence (SEQ ID NO: 1) depicted in FIG. 2 from nucleotide number 52 to 1236.

11. A recombinant vector comprising a nucleotide sequence encoding a cleavage site sandwiched between an .alpha.-N-acetylgalactosaminidase coding sequence and a protein A domain E coding sequence arranged in the same translational reading frame, controlled by a second nucleotide sequence that regulates the expression of a fusion protein in a mammalian host cell.

12. A recombinant vector pAGB-3, as deposited with the Agricultural Research Culture Collection having accession number B-18724.

13. A recombinant vector comprising a nucleotide sequence encoding a cleavage site sandwiched between an .alpha.-N-acetylgalactosaminidase coding sequence and a protein antigen coding sequence arranged in the same translational reading frame, controlled by a second nucleotide sequence that regulates the expression of a fusion protein in a mammalian host cell.

14. The recombinant vector of claim 11 or 13 which further includes a nucleotide sequence encoding a selectable marker.

15. The recombinant vector of claim 11 or 13 in which the cleavage site is a collagenase substrate.

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