Purified telomerose

• US 6,261,556 B1
• Filed: 10/18/1999
• Issued: 07/17/2001
• Est. Priority Date: 08/04/1995
• Status: Expired due to Term

• Current Assignee: Geron Corporation
• Original Assignee: Geron Corporation
• Inventors: Vasserot, Alain P., Kealey, James T., Lichtsteiner, Serge P., Weinrich, Scott L., Pruzan, Ronald A., Atkinson, Edward M. III
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Saidha, Tekchand
• Application Number:
  US09/420,056
• Time in Patent Office: 638 Days
• Field of Search: 424/94.5, 435/194, 435/252.3, 435/320.1, 435/91.3, 536/23.2, 935/8, 935/9, 935/14, 530/412, 530/413
• US Class Current: 424/94.5
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Abstract

This invention provides purified human telomerase and methods of purifying it. The methods involve the use of several sequential steps, including the use of a first matrix that binds molecules bearing negative charges, a matrix that binds molecules bearing positive charges, a second matrix that binds molecules bearing negative charges, an affinity purification step and a matrix that separates molecules according to their size.

Claims

1. A composition comprising human telomerase protein having at least 2,000-fold increased purity compared with a crude extract of cells from adenovirus-transformed kidney cell line (293 cells), which when associated with telomerase RNA component has a molecular weight of 200-2,000 kDa.

2. The composition of claim 1, having between 2,000-fold and 100,000-fold increased purity.

3. The composition of claim 1, having between 3,000-fold and 60,000-fold increased purity.

4. The composition of claim 1, having at least 20,000 fold increased purity.

5. The composition of claim 3, having at least 20,000 fold increased purity.

6. Purified human telomerase core enzyme protein having at least 2,000-fold increased purity compared with a crude extract of cells from adenovirus-transformed kidney cell line (293 cells), which when associated with telomerase RNA component has DNA polymerase activity and a molecular weight of 200-2,000 kDa.

7. The purified protein of claim 6, wherein the core enzyme protein has between 2,000-fold and 100,000-fold increased purity.

8. The purified protein of claim 6, wherein the core enzyme protein has between 3,000-fold and 60,000-fold increased purity.

9. The purified protein of claim 6, wherein the core enzyme protein has at least 20,000 fold increased purity.

10. The purified protein of claim 8, wherein the core enzyme protein has at least 20,000 fold increased purity.

11. A purified cell extract that has measurable telomerase activity in 0.2 μ

g of protein when quantified in a telomere primer elongation assay in which ³²P-labeled primer extensions are separated on a gel and detected using a phosphoimager screen, wherein the telomerase activity elutes with an apparent molecular weight of 200-2,000 kDa when fractionated by gel filtration chromatography.

12. The purified cell extract of claim 11, wherein the telomerase activity is enriched between 3,000-fold and 60,000-fold compared with a crude extract of cells from adenovirus-transformed kidney cell line (293 cells).