Chondroitin lyase enzymes

• US 6,054,569 A
• Filed: 09/17/1997
• Issued: 04/25/2000
• 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:
  US08/931,952
• Time in Patent Office: 951 Days
• Field of Search: 435/232, 435/320.1, 435/252.3, 424/94.5, 536/23.2
• US Class Current: 536/23.2
• 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. An isolated nucleic acid fragment having the sequence of Sequence ID No. 1 or naturally occurring sequences having conservative or degenerative substitutions thereof, encoding chondroitinase AC.

2. The nucleic acid fragment of claim 1 encoding chondroitinase AC having the amino acid sequence of Sequence ID No. 2.

3. The nucleic acid fragment of claim 1 having the sequence of Sequence ID No. 1.

4. An isolated nucleic acid fragment having the sequence of Sequence ID No. 3 or naturally occurring sequences having conservative or degenerative substitutions thereof, encoding chondroitinase B.

5. The nucleic acid fragment of claim 4 encoding chondroitinase B having the amino acid sequence of Sequence ID No. 4.

6. The nucleic acid fragment of claim 4 having the sequence of Sequence ID. No. 3.