Genetically modified cells and methods for expressing recombinant heparanase and methods of purifying same

• US 6,475,763 B1
• Filed: 01/19/2000
• Issued: 11/05/2002
• Est. Priority Date: 09/02/1997
• Status: Expired due to Fees

• Current Assignee: Insight Biopharmaceuticals Limited
• Original Assignee: Insight Strategy & Marketing Ltd.
• Inventors: Pecker, Iris, Ben-Artzi, Hanna, Moskowitz, Haim, Gilboa, Ayelet, Mimon, Madlene, Yacoby-Zeevi, Oron, Miron, Daphna, Shlomi, Yinon, Ayal-Hershkovitz, Maty, Peleg, Yoav
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Rao, Manjunath N.
• Application Number:
  US09/487,716
• Time in Patent Office: 1,021 Days
• Field of Search: 435/4, 435/23, 435/7-91, 435/183, 435/200
• US Class Current: 435/200
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 302/01166 Heparanase (3.2.1.166)
• CPC Class Codes: G01N 2333/988 Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
• CPC Class Codes: G01N 2400/40 Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Bacterial, yeast and animal cells and methods for overexpressing recombinant heparanase in cellular systems, methods of purifying recombinant heparanase therefrom and modified heparanase species which serve as precursors for generating highly active heparanase by proteolysis.

Claims

1. A method of activating a mammalian heparanase protein, the method comprising digesting the mammalian heparanase protein with a protease capable of cleaving said mammalian heparanase protein at a region containing its most hydrophilic sites present within the first 170 N terminal amino acids of said mammalian heparanase protein, as determined using the Kyte-Doolittle method for calculating hydrophilicity, using the Wisconsin University GCG DNA analysis software, so as to release a catalytically active portion of said mammalian heparanase.

2. The method of claim 1, wherein said protease is selected from the group consisting of a cysteine protease, an aspartyl protease, a serine protease and a metalloproteinase.

3. The method of claim 1, wherein said step of digesting the mammalian heparanase protein by said protease is effected at a pH in which said protease is active.

4. The method of claim 1, wherein said step of digesting the mammalian heparanase protein by said protease is effected at a pH in which said protease has an optimal protease activity.

5. A method of activating a modified mammalian heparanase protein, wherein the modified mammalian heparanase protein comprises an introduced protease cleavage recognition sequence at a region containing the most hydrophilic sites present within the first 170 N terminal amino acids of a natural mammalian heparanase protein as determined using the Kyte-Doolittle method for calculating hydrophilicity, using the Wisconsin University GCG DNA analysis software, the method comprising digesting the modified mammalian heparanase protein with a matching protease being capable of cleaving said introduced protease cleavage recognition sequence.

6. The method of claim 5, wherein said protease is selected from the group consisting of a cysteine protease, an aspartyl protease, a serine protease and a metalloproteinase.

7. The method of claim 5, wherein digesting the modified mammalian heparanase protein by said matching protease is effected at a pH in which said protease is active.

8. The method of claim 5, wherein digesting the modified mammalian heparanase protein by said matching protease is effected at a pH in which said protease has an optimal protease activity.