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1. WO2007109697 - UTILISATION DE LA FUSION DE LA PROTÉINE S PERMETTANT UNE SOLUBILISATION PROTÉIQUE

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CLAIMS

What is claimed is:

1. A vector containing a multiple cloning site comprising a nucleic acid sequence encoding a

PrS tag or a PΓST tag from Myxococcus xanthus.
2. The vector of claim 1, wherein the multiple cloning site comprises SEQ ID NO: 1.
3. The vector of claim 2, wherein the multiple cloning site comprises SEQ ID NO: 2 .
4. The vector of claim 1 , wherein the vector is a high expression cold shock vector.
5. The vector of claim 4, wherein the cold shock vector is a pColdIII vector.
6. The vector of claim 4 expressed in Escherichia coli.
7. The vector of claim 1, wherein the vector is a tetracycline-inducible PST expression system.
8. The vector of claim 7 expressed in a mammalian host cell.
9. The vector of claim 8, wherein the mammalian host cell is from a TREx 293 human embryonic kidney cell line.
10. A method for enhancing solubility of a target protein, comprising :
a) fusing a nucleic acid sequence encoding at least one N-terminal domain of Protein S (PrS tag) from Myxococcus xanthus to a nucleic acid sequence encoding the target protein to create a nucleic acid sequence encoding a PrS tagged target protein;

b) positioning the Protein S tagged target protein of step (a) into a vector;

c) transforming a host cell with the vector; and d) culturing the host cell under conditions suitable for gene expression,

whereby the expressed PrS tagged target protein is more soluble than the untagged target protein.

11. The method of claim 10, wherein the nucleic acid sequence encodes a PrS2 tag having two tandemly repeated N-terminal domains of Protein S.

12. The method of claim 10, wherein after performing step (d), the PrS tag is optionally cleaved from the target protein using a protease.

13. The method of claim 10, wherein the host cell is a prokaryote.

14. The method of claim 1, wherein the target protein is from a prokaryote or a eukaryote.

15. The method of claim 1, wherein the vector is an Escherichia coli pCold expression system containing a multiple cloning site comprising the nucleic acid encoding the PrS tagged target protein.

16. The method of claim 1, wherein the host cell is a eukaryote.

17. The method of claim 1, wherein the vector is a mammalian tetracycline-inducible system.

18. The method of claim 1 , wherein the N-terminal domain nucleic acid sequence encodes from 85-100 amino acid residues.

19. The method of claim 1, wherein the N-terminal domain nucleic acid sequence encodes 93 amino acid residues.

20. A PrS-tagged protein molecule bound to one or more myxospores of Myxococcus xanthus.

21. A method for purifying a PrS-tagged or PrS2-tagged protein comprising contacting the tagged protein with an affinity resin comprising myxospores of Myxococcus xanthus, thereby immobilizing the PrS-tagged or PrSi-tagged protein on the affinity resin.

22. The method of claim 21, wherein the PrS-tagged or PrS2-tagged protein is contacted with the affinity resin in the presence of Ca2* or Mg +.

23. The method of claim 22, wherein the PrS-tagged or PrSz-tagged protein is released from the affinity resin by adding an agent that chelates calcium.

24. The method of claim 23, wherein the agent that chelates calcium is EDTA or EGTA.

25. A protein purified according to the method of claim 21.

26. A protein of claim 25. wherein the protein is a PrS- or a PrS2- tagged protein.

27. A kit comprising an affinity resin according to claim 21 and instructions for use in a method of purifying a PrS- or PrS2- tagged protein.

28. A kit according to claim 27, further comprising components for producing a PrS- or PrS2-tagged protein.

29. A method of claim I5 wherein the PrS- or PrS?- tagged protein comprises a linker between the protein S and the target protein.

30. A method of claim 29, wherein the linker is a polypeptide.

31. A method of claim 30, wherein the polypeptide is from 3 to 10 amino acid residues.

32. A method of preparing a target protein for an analytical study, comprising preparing a PrS- or Pr&2-tagged target protein, and performing the analytical study.

33. The method of claim 32, wherein the analytical study is selected from the group consisting of nuclear magnetic resonance spectroscopy, drug screening assays, binding assays and X-ray crystallography or any study requiring a soluble target protein.

34. The method of claim 33, wherein the PrS- or the PrS?-tagged target protein comprises a linker between the PrS tag or PrSa-tag and the target protein.

35. The method of claim 34, wherein the linker is a polypeptide.

36. A method of claim 33, wherein the analytical study is nuclear magnetic resonance spectroscopy.

37. A method of characterizing a target protein comprising fusing at least one PrS tag or a PrS2 tag to the target protein, performing nuclear magnetic resonance spectroscopy and analyzing data from the nuclear magnetic resonance spectroscopy, thereby characterizing the target protein.