Chondroitin lyase enzymes

• US 6,093,563 A
• Filed: 07/08/1994
• Issued: 07/25/2000
• Est. Priority Date: 07/08/1994
• Status: Expired due to Fees

• Current Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Original Assignee: IBEX Technologies R And D, Inc.
• Inventors: Linhardt, Robert, Zimmermann, Joseph, Bennett, D. Clark, Fink, Dominique, Tkalec, Anna Lydia, Laliberte, Maryse, Gu, Kangfu
• Primary Examiner(s): Prouty, Rebecca E.
• Application Number:
  US08/272,247
• Time in Patent Office: 2,209 Days
• Field of Search: 435/232, 424/94.5, 532/23.2
• US Class Current: 435/232
• CPC Class Codes: C12N 9/88 Lyases (4.)

Abstract

The present invention describes a method for the production of two highly purified enzymes capable of degrading chondroitin sulfate polysaccharides. A multi-step purification method incorporating cell disruption, cation exchange chromatography, affinity chromatography, hydroxylapatite chromatography, high resolution ion exchange chromatography and size exclusion is outlined. A 77,000±5,000 Dalton protein capable of degrading chondroitin sulfates A and C and a 55,000±2,300 Dalton protein capable of degrading dermatan sulfate were isolated. The genes encoding these enzymes, chondroitinase AC and chondroitinase B, respectively, have been cloned and methods for their use are described.

Claims

1. A recombinant chondroitinase AC encoded by the nucleotide sequence of Sequence ID No. 1 or naturally occurring sequences having conservative or degenerative substitutions thereof, wherein the chondroitinase AC has been separated from other chondroitinases and has a purity of greater than 99%.

2. The enzyme of claim 1 having the amino acid sequence of Sequence ID No. 2.

3. The chondroitinase of claim 1 having the amino acid sequence shown in Seq. ID No. 2.

4. A recombinant chondroitinase B encoded by the nucleotide sequence of Sequence ID No. 3 or naturally occurring sequences having conservative or degenerative substitutions thereof, wherein the chondroitinase B has been separated from other chondroitinases and has a purity of greater than 99%.

5. The enzyme of claim 4 having the amino acid sequence of Sequence ID No. 4.

6. The chondroitinase of claim 4 having the amino acid sequence shown in Seq. ID No. 4.

7. A chondroitinase B purified to greater than 99% purity from Flavobacterium heparinum, obtainable from the bacteria by a process comprising the steps of:

liberating soluble proteins by disrupting the bacteria;

extracting proteins from the periplasmic space of the disrupted bacteria;

separating the extracted proteins by cation exchange chromatography using a salt or pH gradient;

separating the fractions having enzymatic activity obtained by elution of the cation exchange chromatography matrix by chromatography on a sulfated cellulose resin using a salt or pH gradient;

separating the fractions having enzymatic activity obtained by elution of the sulfated cellulose resin on hydroxyapatite using a salt or pH gradient;

separating the fractions having chondroitinase AC activity from the fractions having chondroitinase B activity by elution of the hydroxyapatite by chromatography using cation exchange chromatography using a salt or pH gradient; and

further purifying the fractions with chondroitinase B activity on the basis of molecular weight.

8. The enzyme of claim 7 where the chondroitinase B has a molecular weight between 52,700 and 57,300 Daltons and is capable of degrading dermatan sulfate or chondroitin sulfate B.

9. The enzyme of claim 7 further comprising a pharmaceutically acceptable carrier.

10. A chondroitinase AC purified to greater than 99% purity from Flavobacterium heparinum, obtainable from the bacteria by a process comprising the steps of:

liberating soluble proteins by disrupting the bacteria;

extracting proteins from the periplasmic space of the disrupted bacteria;

separating the extracted proteins by cation exchange chromatography using a salt or pH gradient;

separating the fractions having enzymatic activity obtained by elution of the cation exchange chromatography matrix by chromatography on a sulfated cellulose resin using a salt or pH gradient;

separating the fractions having enzymatic activity obtained by elution of the sulfated cellulose resin on hydroxyapatite using a salt or pH gradient; and

separating the fractions having chondroitinase AC activity from the fractions having chondroitinase B activity by elution of the hydroxyapatite by chromatography using cation exchange chromatography using a salt or pH gradient.

11. The enzyme of claim 10 further comprising a pharmaceutically acceptable carrier.

12. The enzyme of claim 10 where the chondroitinase AC has a molecular weight between 72,000 and 82,000 Daltons and is capable of degrading chondroitin sulfate A and chondroitin sulfate C.