Engineering of leader peptides for the secretion of recombinant proteins in bacteria

• US 20030180937A1
• Filed: 11/05/2002
• Published: 09/25/2003
• Est. Priority Date: 11/05/2001
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: DeLisa, Matthew, Georgiou, George
• Granted Patent: US 7,419,783 B2
• Time in Patent Office: Days
• US Class Current: 435/252.8
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence

Abstract

The present invention provides methods of isolating of leader peptides capable of directing export of heterologous proteins from the bacterial cytoplasm. The methods rely on the screening of libraries of putative leader peptides or of leader peptide mutants for sequences that allow rapid export and thus can rescue a short-lived reporter protein from degradation in the cytoplasm. The mutant leader peptides identified herein are shown to confer significantly higher steady state levels of export not only for short-lived reporter protein but also for other stable, long-lived proteins. These leader peptides can be used to direct or enhance protein secretion. The present invention further discloses methods for the export of cytoplasmically folded protein via the Tat pathway. Proteins having disulfide bonds are first folded within the cytoplasm in suitable oxidizing mutant strains. Such cytoplasmically pre-folded proteins containing disulfide bonds are then exported via the Tat pathway.

Claims

1. A method of identifying a leader peptide that directs protein export in bacteria, comprising the steps of:

a) obtaining a library of nucleic acid sequences encoding mutated leader peptides;

b) constructing a plurality of expression cassettes comprising said nucleic acid sequences encoding mutated leader peptides upstream of a nucleic acid sequence encoding a short-lived reporter protein, wherein the short lived reporter protein is subject to degradation in the cytoplasm of bacteria;

c) expressing said plurality of expression cassettes in bacteria;

d) measuring expression of said reporter protein in said bacteria; and

e) collecting bacteria cells having increased expression of said reporter protein relative to bacteria that do not express a peptide leader peptide that directs protein export of said short lived reporter protein,

wherein the mutated leader peptide expressed in said cells that have increased expression of said reporter protein is a leader peptide that directs export from the cytoplasm, whereby said export rescues said short-lived reporter protein from degradation in the cytoplasm.

2. The method of claim 1, wherein said short-lived reporter protein is constructed by operably linking a cytoplasmic degradation sequence to the nucleic acid sequence encoding said reporter protein.

3. The method of claim 2, wherein said cytoplasmic degradation sequence is selected from the group consisting of SsrA, PEST, sequences recognized by LON, sequences recognized by ClpAP, sequences recognized by ClpXP, sequences recognized by Stsh and sequences recognized by HslUV.

4. The method of claim 2, wherein said cytoplasmic degradation sequence is attached to the N-terminal or the C-terminal end of said reporter protein.

5. The method of claim 1, wherein said reporter protein is selected from the group consisting of a fluorescent protein, an enzyme, a transport protein, an antibiotic resistance enzyme, a toxin immunity protein, a bacteriophage receptor protein and an antibody.

6. The method of claim 5, wherein said fluorescent protein is green fluorescent protein.

7. The method of claim 1, wherein said nucleic acid sequences encoding mutated leader peptides are generated by a method selected from the group consisting of random mutagenesis, error-prone PCR, site-directed mutagenesis and generation of random DNA fragments.

8. The method of claim 1, wherein said leader peptide mediates protein secretion through a pathway selected from the group consisting of the general secretory (Sec) pathway, the signal recognition particle (SRP)-dependent pathway, the YidC-dependent pathway and the twin-arginine translocation (Tat) pathway.

9. The method of claim 1, further comprising the steps of:

f) cloning a selected nucleic acid sequence encoding a mutated leader peptide from collected bacteria cells having increased expression of said reporter protein compared to bacteria cells that express a wild type leader peptide, and

g) constructing an expression cassette comprising said nucleic acid sequence encoding said mutated selected leader peptide upstream of a nucleic acid sequence encoding a heterologous polypeptide of interest.

10. The method of claim 9, still further comprising expressing said expression cassette in bacteria so that said leader peptide directs increased export of said heterologous polypeptide in bacteria.

11. The method of claim 1, wherein said bacteria are Gram negative bacteria.

12. A method of screening for a compound that inhibits or enhances protein export in bacteria, comprising the steps of:

a) constructing an expression cassette comprising a nucleic acid sequence encoding a mutated leader peptide that directs protein export in bacteria upstream of a nucleic acid sequence encoding a short-lived reporter protein, wherein the short lived reporter protein is subject to degradation in the cytoplasm of bacteria;

b) expressing said expression cassette in bacteria in the presence or absence of said compound; and

c) measuring expression of said reporter protein in said bacteria,

wherein increased expression of said reporter protein measured in the presence of said compound indicates said compound enhances protein export, and wherein decreased expression of said reporter protein measured in the presence of said compound indicates said compound inhibits protein export, whereby protein export rescues said short-lived reporter protein from degradation in the cytoplasm.

13. The method of claim 12, wherein said short-lived reporter protein is constructed by operably linking a cytoplasmic degradation sequence to the nucleic acid sequence encoding said reporter protein.

14. The method of claim 13, wherein said cytoplasmic degradation sequence is selected from the group consisting of SsrA, PEST, sequences recognized by LON, sequences recognized by ClpAP, sequences recognized by ClpXP, sequences recognized by Stsh and sequences recognized by HslUV.

15. The method of claim 13, wherein said cytoplasmic degradation sequence is attached to the N-terminal or the C-terminal of said reporter protein.

16. The method of claim 12, wherein said reporter protein is selected from the group consisting of fluorescent protein, an enzyme, a transport protein, an antibiotic resistance enzyme, a toxin immunity protein, a bacteriophage receptor protein and an antibody.

17. The method of claim 16, wherein said fluorescent protein is green fluorescent protein.

18. The method of claim 12, wherein said bacteria are Gram negative bacteria.

19. A method of identifying a leader peptide that directs increased protein export through the Twin Arginine Translocation pathway, comprising the steps of:

a) generating a library of nucleic acid sequences encoding mutated leader peptides specific for the Twin Arginine Translocation pathway;

b) constructing a plurality of expression cassettes comprising said nucleic acid sequences encoding mutated leader peptides upstream of a nucleic acid sequence encoding a short-lived reporter protein, wherein the short lived reporter protein is subject to degradation in the cytoplasm of bacteria;

c) expressing said expression cassettes in bacteria;

d) measuring the expression of said reporter protein in said bacteria; and

e) collecting bacteria cells having increased expression of said reporter protein relative to bacteria that do not express a peptide leader peptide that directs protein export of said short lived reporter protein,

wherein the mutated leader peptide expressed in said cells that exhibit increased expression of said reporter protein is a leader peptide that directs increased protein export from the cytoplasm through the Twin Arginine Translocation pathway, whereby said export rescues said short-lived reporter protein from degradation in the cytoplasm.

20. The method of claim 19, wherein said short-lived reporter protein is constructed by operably linking a cytoplasmic degradation sequence to the nucleic acid sequence encoding said reporter protein.

21. The method of claim 20, wherein said cytoplasmic degradation sequence is selected from the group consisting of SsrA, PEST, sequences recognized by LON, sequences recognized by ClpAP, sequences recognized by ClpXP, sequences recognized by Stsh and sequences recognized by HslUV.

22. The method of claim 20, wherein said cytoplasmic degradation sequence is attached to the N-terminal or the C-terminal of said reporter protein.

23. The method of claim 19, wherein said reporter protein is selected from the group consisting of fluorescent protein, an enzyme, a transport protein, an antibiotic resistance enzyme, a toxin immunity protein, a bacteriophage receptor protein and an antibody.

24. The method of claim 23, wherein said fluorescent protein is green fluorescent protein.

25. The method of claim 19, wherein said bacteria are Gram negative bacteria.

26. The method of claim 19, wherein said nucleic acid sequences encoding mutated leader peptides specific for the Twin Arginine Translocation pathway are generated by a method selected from the group consisting of random mutagenesis, error-prone PCR, site-directed mutagenesis and generation of random DNA fragments.

27. The method of claim 19, wherein said leader peptide comprises a sequence selected from the group consisting of SEQ ID NOs:

120-128 or a sequence mutated therefrom.

28. A leader peptide that directs increased protein export through the Twin Arginine Translocation pathway prepared by the method of claim 19.

29. An isolated nucleic acid sequence encoding the leader peptide of claim 28.

30. A method of increasing export of heterologous polypeptide through the Twin Arginine Translocation pathway, comprising the steps of:

a) constructing an expression cassette comprising a nucleic acid sequence encoding a leader peptide that directs increased polypeptide export through the Twin Arginine Translocation pathway upstream of a nucleic acid sequence encoding a heterologous polypeptide of interest; and

b) expressing said expression cassette in bacteria so that said leader peptide directs increased export of said heterologous polypeptide through the Twin Arginine Translocation pathway.

31. The method of claim 30, wherein said leader peptide comprises a sequence selected from the group consisting of SEQ ID NOs:

120-128.

32. A method of screening for a compound that inhibits or enhances protein export through the Twin Arginine Translocation pathway, comprising the steps of:

a) constructing an expression cassette comprising a nucleic acid sequence encoding a leader peptide upstream of a nucleic acid sequence encoding a short-lived reporter protein, wherein the short lived reporter protein is subject to degradation in the cytoplasm of bacteria, and wherein said leader peptide directs protein export through the Twin Arginine Translocation pathway;

b) expressing said expression cassette in said bacteria in the presence or absence of said compound; and

c) measuring expression of said reporter protein in said bacteria,

wherein increased expression of said reporter protein measured in the presence of said compound indicates said compound enhances protein export through the Twin Arginine Translocation pathway, and decreased expression of said reporter protein measured in the presence of said compound indicates said compound inhibits protein export through the Twin Arginine Translocation pathway.

33. The method of claim 32, wherein said short-lived reporter protein is constructed by operably linking a cytoplasmic degradation sequence to the nucleic acid sequence encoding said reporter protein.

34. The method of claim 33, wherein said cytoplasmic degradation sequence is selected from the group consisting of SsrA, PEST, sequences recognized by LON, sequences recognized by ClpAP, sequences recognized by ClpXP, sequences recognized by Stsh and sequences recognized by HslUV.

35. The method of claim 33, wherein said cytoplasmic degradation sequence is attached to the N-terminal or the C-terminal of said reporter protein.

36. The method of claim 32, wherein said reporter protein is selected from the group consisting of fluorescent protein, an enzyme, a transport protein, an antibiotic resistance enzyme, a toxin immunity protein, a bacteriophage receptor protein and an antibody.

37. The method of claim 36, wherein said fluorescent protein is green fluorescent protein.

38. A method of producing a biologically-active heterologous polypeptide in a cell, comprising the steps of:

a) constructing an expression cassette comprising a nucleic acid sequence encoding a leader peptide that directs protein export through the Twin Arginine Translocation pathway upstream of a nucleic acid sequence encoding said heterologous polypeptide; and

b) expressing said expression cassette in a bacterial cell, wherein said heterologous polypeptide is produced in a biologically-active form.

39. The method of claim 38, wherein the heterologous polypeptide comprises and antibody fragment.

40. The method of claim 38, wherein said leader peptide comprises a sequence selected from the group consisting of SEQ ID NOs:

25-46 and 120-128.

41. The method of claim 38, wherein said heterologous polypeptide is selected from the group consisting of a polypeptide secreted from said bacterial cell, a polypeptide isolatable from the periplasm of said bacterial cell, an integral membrane protein and a polypeptide isolatable from the culture supernatant of said bacterial cell.

42. The method of claim 38, wherein said heterologous polypeptide is a mammalian polypeptide.

43. The method of claim 42, wherein said mammalian polypeptide is selected from the group consisting of tissue plasminogen activator, pancreatic trypsin inhibitor, an antibody, an antibody fragment and a toxin immunity protein.

44. The method of claim 38, wherein said heterologous polypeptide is selected from the group consisting of a polypeptide in native conformation, a mutated polypeptide and a truncated polypeptide.

45. The method of claim 38, wherein said bacterial cell has an oxidizing cytoplasm.

46. The method of claim 38, wherein said heterologous polypeptide forms disulfide bonds in the cytoplasm of said bacterial cell

47. The method of claim 38, wherein a second heterologous polypeptide is expressed in the cytoplasm of said bacteria and associates in said cytoplasm with the heterologous polypeptide, wherein the second heterologous polypeptide lacks said leader peptide and wherein said leader peptide directs export of said second heterologous polypeptide associated with said heterologous polypeptide by protein export through the Twin Arginine Translocation pathway,

48. The method of claim 45, wherein said bacterial cell is a Gram negative bacteria.

49. The method of claim 45, wherein said bacterial cell is selected from the group consisting of an E. coli trxB mutant, E. coli gor mutant and E. coli trxB gor double mutant.

50. The method of claim 45, wherein said cell secretes at least one biologically-active heterologous polypeptide containing from about 2 to about 17 disulfide bonds.

51. The method of claim 50, wherein two of said heterologous polypeptides are linked by at least one disulfide bond.

52. An isolated leader peptide that directs protein secretion and export through the Twin Arginine Translocation pathway.

53. The isolated leader peptide of claim 52, wherein said leader peptide comprises a sequence selected from the group consisting of SEQ ID NOs:

25-46 and 120-128.

54. A recombinant nucleic acid sequence encoding a leader peptide selected from the group consisting of SEQ ID NOs:

25-46 and 120-128.