Methods for Enhancing Bacterial Cell Display of Proteins and Peptides
First Claim
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1. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and native C-terminus of OmpX, wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus, and wherein the linker is 3-8 residues in length and comprises a glycine and one or more basic amino acids.
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Abstract
Methods of making and using bacterial display polypeptide libraries using circularly permuted OmpX (CPX) variants are disclosed. The invention further relates to methods for enhancing the display of proteins and peptides at the surface of bacteria by optimizing linkers and incorporating mutations at positions 165 and 166 of CPX.
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Citations
52 Claims
- 1. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and native C-terminus of OmpX, wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus, and wherein the linker is 3-8 residues in length and comprises a glycine and one or more basic amino acids.
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10. (canceled)
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19-21. -21. (canceled)
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26-30. -30. (canceled)
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34-36. -36. (canceled)
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38-39. -39. (canceled)
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41-42. -42. (canceled)
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45. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and native C-terminus of OmpX, wherein the linker is 3-8 residues in length and comprises a glycine and one or more basic amino acids, and
wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus and one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein consisting of SEQ ID NO: - 1.
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46. (canceled)
- 47. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and native C-terminus of OmpX, wherein the linker is 3-8 residues in length, and wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus and one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein consisting of SEQ ID NO:
- 49. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and native C-terminus of OmpX, wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus, and wherein the linker is 3-8 residues in length, the linker comprises one or more basic amino acids, the first residue of the linker is a glycine, and the third residue of the linker is selected from the group consisting of arginine, lysine, serine, histidine, glutamine, and asparagine.
Specification