Prokaryotic expression of soluble, active Dkk
Dickkopf (Dkk) proteins inhibit the canonical Wnt signaling pathway. Each of the members of the Dkk family has been previously cloned and expressed as a soluble protein in eukaryotic cells, while expression in bacterial cells has resulted in the formation of insoluble inclusion bodies that require further processing. The present invention provides compositions and methods for producing soluble, active dkk protein in prokaryotic host cells, by expressing the dkk protein as a fusion protein with a solubilization molecule, thereby providing an inexpensive and convenient source of pure active Dkk.
1. A method for producing soluble, active Dickkopf (Dkk) protein, or a portion thereof, comprising
a) growing a culture of prokaryotic host cells that express all or a portion of a Dkk protein as a fusion protein encoded by an expression construct, wherein said fusion protein comprises all or a portion of a Dkk protein fused to a solubilization molecule;
b) isolating said prokaryotic host cells;
c) lysing said prokaryotic host cells;
d) isolating the soluble portion of said prokaryotic cell lysate; and
e) purifying said fusion protein from said soluble portion thereby producing soluble, active Dkk protein, or a portion thereof.
2. The method of claim 1 , wherein step (a) includes adding isopropyl-1-thio-β-D-galactoside to the culture of prokaryotic host cells.
3. The method of claim 1 , wherein the fusion protein further comprises a protein purification tag.
4. The method of claim 3 , wherein the fusion protein further comprises one or more cleavage sequences located between the protein purification tag or solubilization molecule and the Dkk protein.
5. The method of claim 1 , wherein the expression construct comprises a portion of the Dkk coding sequence and said portion comprises only one cysteine-rich domain.
6. The method of claim 5 , wherein said cysteine-rich domain is a carboxy-terminal cysteine-rich domain.
7. The method of claim 1 , wherein said expression construct is derived from a prokaryotic expression vector.
8. The method of claim 7 , wherein said prokaryotic expression vector is pET32a.
9. The method of claim 1 , wherein said Dkk protein is selected from the group consisting of Dkk-1, Dkk-2, Dkk-3 and Dkk-4.
10. The method of claim 9 , wherein said Dkk protein is Dkk-2.
11. The method of claim 1 , wherein said prokaryotic host cells have a mutation in a trxB gene, a gor gene or both said trxB gene and said gor gene.
12. The method of claim 11 , wherein said prokaryotic host cells further have a mutation in a lacy1* gene.
13. The method of claim 1 , wherein the isolation step (d) is carried out by centrifugation.
14. The method of claim 1 , wherein said purification step (e) comprises the use of a metal chelate column.
15. The method of claim 1 , wherein said purification step (e) comprises an HPLC step.
16. An expression construct comprising nucleic acids encoding all or a portion of Dickkopf (Dkk)-2 as a fusion protein, wherein said fusion protein comprises all or a portion of a Dkk protein fused to a solubilization molecule.
17. The expression construct of claim 16 , further comprising nucleic acids encoding a protein purification tag.
18. The expression construct of claim 16 , wherein said Dkk-2 protein is soluble when expressed in a prokaryotic host cell.
19. A prokaryotic host cell comprising an expression construct encoding a portion of a Dkk protein fused to a solubilization molecule, wherein said portion comprises only one cysteine-rich domain of the Dkk protein.
20. The prokaryotic host cell of claim 19 , wherein the Dkk protein is the Dkk-2 protein.
21. The prokaryotic host cell of claim 19 , wherein said cysteine-rich domain is the C-terminal cysteine-rich domain of the Dkk protein.
22. The prokaryotic host cell of claim 19 , wherein the Dkk protein is soluble when expressed in the prokaryotic host cell.
23. A prokaryotic host cell comprising an expression construct encoding all or a portion of Dkk-2 protein fused to a solubilization molecule.
24. The prokaryotic host cell of claim 23 , wherein said prokaryotic host cell has a mutation in a trxB gene, a gor gene or both said trxB gene and said gor gene.