Nucleic acid encoding site specific recombinase and nuclear receptor fusion protein

• US 6,040,430 A
• Filed: 12/26/1995
• Issued: 03/21/2000
• Est. Priority Date: 06/28/1993
• Status: Expired due to Term

• Current Assignee: European Molecular Biology Laboratory
• Original Assignee: European Molecular Biology Laboratory
• Inventors: Stewart, Francis
• Primary Examiner(s): Walsh, Stephen
• Assistant Examiner(s): PAK, MICHAEL D
• Application Number:
  US08/564,264
• Time in Patent Office: 1,547 Days
• Field of Search: 530/350, 530/358, 435/7.1, 435/69.1, 435/320.1, 435/240.2, 435/325, 435/6, 435/69.7, 435/195, 536/23.1, 536/23.2, 536/23.3, 536/23.5
• US Class Current: 530/358
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

An invention is described that permits the regulation of recombination in cells, organisms or appropriate cell-free systems. The invention involves creating fusion proteins between recombinase proteins, or components of recombinase systems, and ligand binding domains derived from nuclear receptors. The fusion proteins show little recombinase activity in the absence of the ligand that binds to the ligand binding domain. Upon binding of the ligand, recombinase activity is induced. The invention provides a practical means to regulate recombination in cells and organisms and, by linking ligand binding to recombination, provides a simple means whereby ligand binding can be measured as recombination achieved.

Claims

1. A fusion protein, comprising (1) a site specific recombinase protein and (2) the ligand binding domain of a nuclear receptor which is attached at the C- or N-terminus of said site specific recombinase protein, such that, in a cell or cell-free system containing DNA target sites which are specifically recognized by said site specific recombinase protein:

(a) site specific recombinase activity of said fusion protein in relation to the DNA target sites is inhibited in the absence of ligand binding to said ligand binding domain, and

(b) inhibition of said site specific recombinase activity of said fusion protein in relation to the DNA target sites is relieved by binding of ligand to said ligand binding domain.

2. The fusion protein of claim 1, wherein the site specific recombinase protein is a specific DNA binding component of a site specific recombinase complex.

3. The fusion protein of claim 1, wherein the site specific recombinase protein is a recombinase enzyme component of a site specific recombinase complex.

4. The fusion protein of claim 1, wherein the nuclear receptor is selected from the group consisting of a vertebrate glucocorticoid, estrogen, progesterone and androgen receptor.

5. The fusion protein of claim 1, wherein the site specific recombinase protein is selected from the group consisting of FLP recombinase, Cre recombinase, R recombinase from the Zygosaccharomyces rouxii plasmid pSR1, A recombinase from the Kluyveromyces drosophilarium plasmid pKD1, A recombinase from the Kluyveromyces waltii plasmid pKW1, the specific DNA binding component of the λ

Int recombinase complex and the enzyme component of the λ

Int recombinase complex.

6. A nucleic acid which encodes the fusion protein of claim 1.

7. A recombinant vector, comprising at least one copy of the nucleic acid of claim 6.

9. A cell containing the nucleic acid of claim 6.

10. A cell containing the recombinant vector of claim 7.

11. A method for determining the binding of a ligand to be evaluated to the ligand binding domain of a nuclear receptor, comprising

(a) introducing the fusion protein of claim 1 into a cell or appropriate cell-free system containing DNA target sites which are specifically recognized by the site specific recombinase protein,

(b) if the cell or cell-free system does not already contain the ligand to be evaluated, introducing the ligand or a sample suspected of containing the ligand into the cell or cell-free system;

and

(c) determining the binding of the ligand to the ligand binding domain by detecting the presence of or change in recombinase activity of the fusion protein in relation to the DNA target sites.

12. The method of claim 11, wherein the ligand binding domain of the fusion protein is a mutation of a wild-type ligand binding domain.

13. A method for determining the binding of a ligand to be evaluated to the ligand binding domain of a nuclear receptor, comprising

(a) introducing the nucleic acid of claim 6 into a cell or appropriate cell-free system containing DNA target sites which are specifically recognized by the site specific recombinase protein and expressing the nucleic acid to produce the fusion protein;

(b) if the cell or cell-free system does not already contain the ligand to be evaluated, introducing the ligand or a sample suspected of containing the ligand into the cell or cell-free system; and

(c) determining the binding of the ligand to the ligand binding domain by detecting the presence of or change in recombinase activity of the fusion protein in relation to the DNA target sites.

14. The method of claim 13, wherein the ligand binding domain of the fusion protein is mutated relative to the wild-type ligand binding domain.

15. A method for regulating the recombination of DNA target sites, comprising

(a) providing a cell or appropriate cell-free system containing DNA target sites which are specifically recognized by the site specific recombinase protein;

(b) contacting the fusion protein of claim 1 with a ligand for the ligand binding domain of the fusion protein and contacting the DNA target sites with the fusion protein; and

(c) effecting recombination of the DNA target sites by the site specific recombinase protein.

8. The plasmid pHFE1 (DSM 9265).