Isolated CEL II endonuclease

• US 7,129,075 B2
• Filed: 10/17/2003
• Issued: 10/31/2006
• Est. Priority Date: 10/18/2002
• Status: Expired due to Fees

• Current Assignee: Transgenomic, Inc. (Precipio, Inc.)
• Original Assignee: Transgenomic, Inc. (Precipio, Inc.)
• Inventors: D'Alessio, James M., Gerard, Gary F., Shandilya, Harini, Qiu, Peter
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US10/688,665
• Publication Number:
  US 20040166510A1
• Time in Patent Office: 1,110 Days
• Field of Search: None
• US Class Current: 435/199
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The present invention relates to the isolation and characterization of CEL I and CEL II endonuclease proteins. Methods and kits for identifying mismatches in double-stranded DNA are also provided.

Claims

1. A purified and isolated CEL II endonuclease protein having a specific activity of greater than 10,000,000 units per mg protein as determined by DNA solubilization at pH 8.5 or CEL I endonuclease protein having a specific activity of greater than 96,000,000 units per mg protein as determined by DNA solubilization at pH 5.5.

2. The isolated CEL II endonuclease of claim 1, wherein said CEL II endonuclease is substantially free of CEL I endonuclease.

3. The isolated CEL I endonuclease of claim 1, wherein said CEL I endonuclease is substantially free of CEL II endonuclease.

4. A method for preparing an isolated CEL I or CEL II endonuclease protein of claim 1 comprising:

a) extracting proteins from a sample;

b) separating the proteins by concanavalin A affinity chromatography;

c) separating the product of step (b) by anion exchange chromatography; and

d) separating the product of step (c) by Heparin affinity chromatography so that a CEL I or CEL II protein is isolated.

5. The method of claim 4, further comprising separating the product of step (d) by one or more Heparin affinity chromatography steps.

6. The method of claim 4, wherein the sample is of plant origin.

7. The method of claim 6, wherein the plant is celery.

9. A composition comprising the isolated CEL II or CEL I endonuclease of claim 1.

8. A method for separating CEL I and CEL II in a mixture comprising subjecting a mixture containing CEL I and CEL II to heparin affinity chromatography so that CEL I and CEL II are separated.