Secretion of proteins with multiple disulfide bonds in bacteria and uses thereof
First Claim
1. A bacteria genetically transformed with an expression cassette comprising a leader peptide that directs protein export through the Twin Arginine Translocation pathway upstream of a gene encoding a heterologous polypeptide, and wherein the heterologous polypeptide is produced by the bacterial cell and comprises at least one disulfide bond.
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Abstract
The invention provides methods for using the Twin Arginine Translocation pathway in bacteria to produce heterologous polypeptides that have multiple disulfide bonds. Methods of screening polypeptide libraries produced by secretion through the TAT pathway are also provided. The invention provides improved methods for production of heterologous polypeptides having at least one disulfide bond.
32 Citations
41 Claims
- 1. A bacteria genetically transformed with an expression cassette comprising a leader peptide that directs protein export through the Twin Arginine Translocation pathway upstream of a gene encoding a heterologous polypeptide, and wherein the heterologous polypeptide is produced by the bacterial cell and comprises at least one disulfide bond.
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20. A method of producing at least one heterologous polypeptide comprising at least one disulfide bond in a bacterial cell, comprising the steps of:
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a) constructing an expression cassette comprising a leader peptide that directs protein export through the Twin Arginine Translocation pathway upstream of a gene encoding a heterologous polypeptide; and
b) expressing said expression cassette in a bacterial cell comprising an oxidizing cytoplasm, wherein said heterologous polypeptide is produced and comprises at least one disulfide bond. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A method of identifying a nucleic acid encoding a mutated polypeptide that can reconstitute protein oxidation in a secretory compartment, comprising the steps of:
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a) obtaining expression cassettes comprising a leader peptide specific for the Twin Arginine Translocation pathway upstream of nucleic acid sequences encoding mutated polypeptides of a protein with oxidizing activity;
b) expressing said expression cassettes in bacteria that have oxidizing cytoplasm and impaired periplasmic disulfide bond formation; and
c) selecting at least a first bacteria in which said impaired periplasmic disulfide bond formation activity has been complemented by expression of said expression cassette to identify a nucleic acid sequence encoding a mutated polypeptide that can reconstitute protein oxidation in a secretory compartment.
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36. A method of screening a combinatorial library, comprising the steps of:
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a) generating a library of polypeptides of interest;
b) constructing expression cassettes that place a leader peptide specific for the Twin Arginine Translocation pathway upstream of a gene encoding said polypeptides;
c) expressing said expression cassettes in bacteria comprising an oxidizing cytoplasm; and
d) screening for expressed secreted polypeptides. - View Dependent Claims (37, 38, 39, 40, 41)
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Specification