Engineering of leader peptides for the secretion of recombinant proteins in bacteria
First Claim
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.
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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.
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Citations
54 Claims
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1. A method of identifying a leader peptide that directs protein export in bacteria, comprising the steps of:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of screening for a compound that inhibits or enhances protein export in bacteria, comprising the steps of:
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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. - View Dependent Claims (13, 14, 15, 16, 17, 18)
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19. A method of identifying a leader peptide that directs increased protein export through the Twin Arginine Translocation pathway, comprising the steps of:
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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. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method of increasing export of heterologous polypeptide through the Twin Arginine Translocation pathway, comprising the steps of:
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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. - View Dependent Claims (31)
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32. A method of screening for a compound that inhibits or enhances protein export through the Twin Arginine Translocation pathway, comprising the steps of:
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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. - View Dependent Claims (33, 34, 35, 36, 37)
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38. A method of producing a biologically-active heterologous polypeptide in a cell, comprising the steps of:
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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. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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- 52. An isolated leader peptide that directs protein secretion and export through the Twin Arginine Translocation pathway.
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54. A recombinant nucleic acid sequence encoding a leader peptide selected from the group consisting of SEQ ID NOs:
- 25-46 and 120-128.
Specification