Pregnane X receptor compositions, crystals and uses thereof

1. An isolated fusion polypeptide comprising: human pregnane X receptor (PXR) polypeptide or a ligand binding domain thereof (PXR-LBD), fused by a linker polypeptide, to human SRC-1 polypeptide or a fragment thereof, wherein said fragment consists of amino acids 678-700 (SEQ ID NO:38) or 682-700 (SEQ ID NO:35) of the human SRC-1 polypeptide of SEQ ID NO:3, and wherein the fusion polypeptide exhibits increased stability compared to native PXR polypeptide or PXR-LBD.

2. The polypeptide of claim 1 wherein the pregnane X receptor polypeptide or ligand binding domain thereof (PXR-LBD) comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2, respectively.

3. The polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 26.

4. The polypeptide of claim 1 wherein the polypeptide is complexed to a member selected from the group consisting of SR12813, hyperforin, clotrimazole, ritonavir, sulfopyrole, pregnane-16α-carbonitrile, RU-486, rifampicin, dexamethasone, paclitaxel, metyrapone, bisphenol A, diethylhexylphthalate, nonylphenol, phthalate, cisplatin, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, 17β-estradiol (estradiol), pregnenolone (5-pregneno-3β-ol-20-one), progesterone, and Medroxyprogesterone-acetate (MPA).

5. A composition comprising the polypeptide of claim 1 , wherein said polypeptide is at least about 95% pure.

6. A composition comprising the fusion polypeptide of claim 1 .

7. An isolated polynucleotide encoding the fusion polypeptide of claim 1 .

8. The polynucleotide of claim 7 encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 26.

9. A bicistronic cassette comprising the polynucleotide of claim 7 .

10. The bicistronic cassette of claim 9 , wherein the polynucleotide is operably linked to a promoter.

11. An isolated fusion polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 24 or 26.

12. An isolated polynucleotide encoding the polypeptide of claim 11 .

13. The polynucleotide of claim 12 consisting of the nucleotide sequence set forth in SEQ ID NO: 25.

14. The isolated fusion polypeptide of claim 1 , produced by a purification method comprising:

(1) expressing the polypeptide in a bacterial cell;

(2) lysing the cell in an aqueous lysis buffer comprising 25 mM buffer, pH 7.9, 5% Glycerol v/v, 150 mM ionic salt, 1 mM reducing agent and 10 mM Imidazole;

(3) contacting a soluble fraction of the lysed cells with a metal chelate chromatography resin;

(4) washing the resin with said lysis buffer;

(5) eluting the polypeptide from the resin with an aqueous elution buffer comprising 25 mM buffer, pH 7.9, 5% Glycerol v/v, 150 mM ionic salt, 1 mM reducing agent and 250 mM Imidazole;

(6) contacting the polypeptide with a strong anion exchange resin equilibrated with an aqueous buffer A comprising 25 mM buffer, pH 7.9, 5% Glycerol v/v, 150 mM ionic salt and 5 mM reducing agent;

(7) collecting the polypeptide in a flow-through fraction from said resin;

(8) concentrating the polypeptide to a concentration of from about 10 to 20 mg/ml;

(9) adding the polypeptide to a size exclusion column equilibrated with said buffer A;

(10) collecting the polypeptide from the size exclusion column; and, optionally,

(11) freezing the polypeptide in liquid nitrogen.

15. The isolated fusion polypeptide of claim 14 , wherein the bacterial cell is E. coli ; the buffer is Hepes; the ionic salt is NaCl; the reducing agent is DTT; and/or the metal chelate chromatography resin comprises Ni 2+ .

16. The polypeptide of claim 1 , wherein the linker polypeptide is from about 8 to about 13 amino acids in length.