Poly zinc finger proteins with improved linkers

• US 6,479,626 B1
• Filed: 03/01/1999
• Issued: 11/12/2002
• Est. Priority Date: 03/02/1998
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Pabo, Carl O., Kim, Jin-Soo
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US09/260,629
• Time in Patent Office: 1,352 Days
• Field of Search: 530/300, 530/324, 530/345, 530/350, 435/69.7, 514/2, 514/12, 514/21
• US Class Current: 530/300
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Provided herein are chimeric zinc finger proteins comprising flexible linkers of at least 6 amino acids in length. Also provided are methods of making and using these chimeric zinc finger proteins.

Claims

1. A chimeric zinc finger protein that binds to adjacent target sites, the chimeric zinc finger protein comprising:

(i) a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises an engineered zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent; and

(ii) a flexible linker that is six or more amino acids in length;

wherein the first and second domains are fused with the flexible linker.

2. The chimeric zinc finger protein of claim 1, wherein the adjacent target sites are separated by one nucleotide and the flexible linker is seven, eight, or nine amino acids in length.

3. The chimeric zinc finger protein of claim 2, wherein the flexible linker has the amino acid sequence RQKDGERP (SEQ ID NO:

14).

4. The chimeric zinc finger protein of claim 1, wherein the adjacent target sites are separated by two nucleotides and the flexible linker is ten, eleven, or twelve amino acids in length.

5. The chimeric zinc finger protein of claim 4, wherein the flexible linker has the amino acid sequence RQKDGGGSERP (SEQ ID NO:

15).

6. The chimeric zinc finger protein of claim 1, wherein the adjacent target sites are separated by three nucleotides and the flexible linker is twelve or more amino acids in length.

7. The chimeric zinc finger protein of claim 1, wherein the first and the second domains are zinc finger polypeptides.

8. The chimeric zinc finger protein of claim 7, wherein the zinc finger polypeptide is selected from the group consisting of Zif268 and NRE.

9. The chimeric zinc finger protein of claim 7, wherein the zinc finger polypeptides are heterologous.

10. The chimeric zinc finger protein of claim 7, wherein the chimeric zinc finger protein has femtomolar affinity for the adjacent target sites.

11. The chimeric zinc finger protein of claim 10, wherein the chimeric zinc finger protein has about 2-4 femtomolar affinity for the adjacent target sites.

12. The chimeric zinc finger protein of claim 1, wherein the chimeric zinc finger protein further comprises a regulatory domain polypeptide.

13. The chimeric zinc finger protein of claim 1, wherein the first domain comprises a poly-finger zinc finger protein.

14. The chimeric zinc finger protein of claim 13, wherein the second domain comprises a poly-finger zinc finger protein.