Poly zinc finger proteins with improved linkers

• US 6,903,185 B2
• Filed: 05/13/2002
• Issued: 06/07/2005
• 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:
  US10/146,221
• Publication Number:
  US 20020173006A1
• Time in Patent Office: 1,121 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

Chimeric proteins, and methods for their production and use are disclosed. The chimeric proteins comprise a flexible linker between two zinc finger DNA-binding domains, wherein the linker comprises eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first domain and the first conserved cysteine residue of the amino-terminal zinc finger of the second domain.

Claims

1. A DNA-binding protein that binds to adjacent first and second target sites, wherein the adjacent target sites are separated by one nucleotide, the protein comprising first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues;

and

and the first and second ZFPs are joined by an amino acid sequence of 9, 10 or 11 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP.

2. The protein of claim 1, wherein the amino acid sequence joining the first and second ZFPs comprises the amino acid sequence RQKDGERP (SEQ ID NO:

14).

3. A DNA-binding protein that binds to adjacent first and second target sites, wherein the adjacent target sites are separated by two nucleotides, the protein comprising first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues;

and

the first and second ZFPs are joined by an amino acid sequence of 12, 13 or 14 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP.

4. A DNA-binding protein that binds to adjacent first and second target sites, the protein comprising first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues;

and

the first and second ZFPs are joined by an amino acid sequence comprising eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP, wherein the zinc finger polypeptide is selected from the group consisting of Zif268 and NRE.

5. A DNA-binding protein that binds to adjacent first and second target sites, the protein comprising

a regulatory domain;

and

first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues; and

the first and second ZEPs are joined by an amino acid sequence comprising eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP.

6. A DNA-binding protein that binds to first and second adjacent target sites, wherein the adjacent target sites are separated by one nucleotide, the protein comprising:

first and second zinc finger polypeptides (ZFPs), wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by a sequence of 9, 10 or 11 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger.

7. The protein of claim 6, wherein the amino acid sequence joining the first and second ZFPs comprises the amino acid sequence RQKDGERP (SEQ ID NO:

14).

8. A DNA-binding protein that binds to first and second adjacent target sites, wherein the adjacent target sites are separated by two nucleotides, the protein comprising:

first and second zinc finger polypeptides (ZFPs) wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by a sequence of 12, 13 or 14 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger.

9. A DNA-binding protein that binds to first and second adjacent target sites, the protein comprising:

first and second zinc finger polypeptides (ZFPs), wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by an amino acid sequence comprising eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second input ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger,

wherein the zinc finger polypeptide is selected from the group consisting of Zif268 and NRE.

10. A DNA-binding protein that binds to first and second adjacent target sites, the protein comprising:

a regulatory domain polypeptide; and

first and second zinc finger polypeptides (ZFPs), wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by an amino acid sequence comprising eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger.