Isolated CEL II endonuclease
View Patent ↗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.
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.
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.
9. A composition comprising the isolated CEL II or CEL I endonuclease of claim 1 .