Compositions and methods for peptide expression and purification using a type III secretion system

Patent metadata

Field	Value
Publication number	US-9630997-B2
Application number	US-201314404919-A
Country	US
Kind code	B2
Filing date	May 30, 2013
Priority date	May 30, 2012
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

Disclosed are compositions and methods for expressing and purifying a peptide of interest using a Flagellar Type III secretion system. Disclosed are nucleic acid sequences that contain a FlgM nucleic acid sequence, a cleavage site, and a nucleic acid sequence of interest. Also disclosed are polypeptides that contain FlgM, a cleavage site and a peptide of interest. Methods of producing polypeptides that have FlgM, a cleavage site and a peptide of interest are provided.

First claim

We claim:

1. A vector comprising a FlgM nucleic acid sequence operably linked to a nucleic acid sequence encoding a purification tag, a cleavage site, a nucleic acid sequence of interest, and a transcription control element (TCE), wherein the TCE is heterologous to the FlgM nucleic acid sequence and wherein the 5′ to 3′ order of: the sequences is the FlgM nucleic acid sequence, the nucleic acid sequence encoding a purification tag, the cleavage site, and the nucleic acid sequence of interest.

2. The vector of claim 1 , wherein the purification tag comprises poly-histidine, glutathione S-transferase (GST), Myc, HA, FLAG, or maltose binding protein (MBP).

3. The vector of claim 2 , wherein the purification tag is poly-histidine.

4. The vector of claim 1 , wherein the FlgM nucleic acid sequence is wild type FlgM.

5. The vector of claim 1 , wherein the cleavage site comprises a Tobacco Etch Virus (TEV) protease cleavage site or an Enterokinase (ETK) cleavage site.

6. The vector of claim 1 , wherein the nucleic acid sequence of interest encodes a cysteine-rich peptide.

7. The vector of claim 6 , wherein the cysteine-rich peptide comprises a neuroactive toxin.

8. The vector of claim 1 , wherein the TCE is a constitutive TCE or a regulatable TCE.

9. The vector of claim 8 , wherein the regulatable TCE comprises an inducible promoter.

10. The vector of claim 9 , wherein the inducible promoter comprises a P araBAD promoter.

11. A bacterial host cell comprising the vector of claim 1 .

12. The bacterial host cell of claim 11 , wherein the FlgM nucleic acid sequence, the purification tag nucleic acid sequence, the cleavage site, the nucleic acid sequence of interest, and the TCE have inserted into the bacterial host cell in the 5′ to 3′ order of: the FlgM nucleic acid sequence, the nucleic acid sequence encoding a purification tag, the cleavage site, and the nucleic acid sequence of interest.

13. The bacterial host cell of claim 11 , that is a Salmonella enterica or an Escherichia coli cell.

14. A polypeptide comprising a FlgM peptide operably fused to a cleavage site, a protein of interest, and a purification tag in the N- to C-terminus order of the FlgM peptide, the purification tag, the cleavage site, and the protein of interest.

15. The polypeptide of claim 14 , wherein the purification tag comprises poly-histidine.

16. A method of producing a peptide of interest comprising:
(a) culturing a bacterial host cell in culture media, wherein the bacterial host cell comprises a FlgM nucleic acid sequence operably linked to a nucleic acid sequence encoding a purification tag, a cleavage site, a nucleic acid sequence of interest, and a transcription control element (TCE), wherein the TCE is heterologous to the FlgM nucleic acid sequence and wherein the 5′ to 3′ order of the sequences in the genome is the FlgM nucleic acid sequence, the nucleic acid sequence encoding a purification tag, the cleavage site, and the nucleic acid sequence of interest.

17. The method of claim 16 , wherein the purification tag comprises poly-histidine, glutathione S-transferase (GST), Myc, HA, FLAG, or maltose binding protein (MBP).

18. The method of claim 17 , wherein the purification tag is poly-histidine.

19. The method of claim 16 , wherein the FlgM nucleic acid sequence is wild type FlgM.

20. The method of claim 16 , wherein the cleavage site comprises a Tobacco Etch Virus (TEV) protease cleavage site or an Enterokinase (ETK) cleavage site.

21. The method of claim 16 , wherein the nucleic acid sequence of interest encodes a cysteine-rich peptide.

22. The method of claim 21 , wherein the cysteine-rich peptide is a neuroactive toxin.

23. The method of claim 16 , wherein the TCE is a constitutive TCE or a regulatable TCE.

24. The method of claim 16 , wherein the regulatable TCE comprises an inducible promoter.

25. The method of claim 24 , wherein the inducible promoter comprises a P araBAD promoter.

26. The method of claim 16 , wherein the bacterial host cell is a Salmonella enterica or an Escherichia coli cell.

27. The method of claim 16 , further comprising: (b) purifying the peptide of interest from the culture media.

28. The method of claim 27 , wherein step (b) comprises use of an affinity column.

29. The method of claim 28 , wherein the affinity column is a σ 28 affinity column.

30. The method of claim 16 , wherein the bacterial host cell is cultured in media comprising about 200 mM to about 400 mM NaCl or KCl and wherein more of the peptide of interest is produced as compared to bacterial host cells cultured in media comprising 100 mM NaCl or KCl, respectively.

Assignees

• Univ Utah Res Found

Inventors

• Hughes Kelly T
• Olivera Baldomero M

Classifications

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• C07K14/255

  Salmonella (G) · CPC title

• C07K14/43522

  from scorpions · CPC title