Polypeptide display libraries and methods of making and using thereof
First Claim
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1. A composition comprising:
- a host cell, wherein the host cell is a bacterial cell; and
a fusion protein disposed in an outer membrane of the host cell, the fusion protein comprising(a) a circularly permuted bacterial outer membrane protein, comprising a bacterial OmpX protein or a bacterial OmpA protein, wherein(i) the native N-terminus and the native C-terminus of the bacterial OmpX protein or the bacterial OmpA protein are joined by a linker, wherein the linker is a short flexible peptide, and(ii) a non-native N-terminus and a non-native C-terminus are derived from an extracellular loop of the bacterial OmpX protein or the bacterial OmpA protein, wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly, and(b) a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus of the circularly permuted bacterial outer membrane protein, wherein the passenger polypeptide is displayed on the outer membrane of the host cell.
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Abstract
Disclosed herein are expression vectors which display a passenger polypeptide on the outer surface of a biological entity. As disclosed herein the displayed passenger polypeptide is capable of interacting or binding with a given ligand. Also disclosed are methods of making and using the expression vectors. N/C terminal fusion expression vectors and methods of making and using are also disclosed.
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Citations
21 Claims
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1. A composition comprising:
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a host cell, wherein the host cell is a bacterial cell; and a fusion protein disposed in an outer membrane of the host cell, the fusion protein comprising (a) a circularly permuted bacterial outer membrane protein, comprising a bacterial OmpX protein or a bacterial OmpA protein, wherein (i) the native N-terminus and the native C-terminus of the bacterial OmpX protein or the bacterial OmpA protein are joined by a linker, wherein the linker is a short flexible peptide, and (ii) a non-native N-terminus and a non-native C-terminus are derived from an extracellular loop of the bacterial OmpX protein or the bacterial OmpA protein, wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly, and (b) a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus of the circularly permuted bacterial outer membrane protein, wherein the passenger polypeptide is displayed on the outer membrane of the host cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 16, 17, 19, 20)
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10. A polypeptide display library comprising:
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a plurality of host cells, wherein the host cells are bacterial cells;
each member of the plurality comprisinga fusion protein disposed in an outer membrane of the host cell, the fusion protein comprising (a) a circularly permuted bacterial outer membrane protein, comprising a bacterial OmpX protein or a bacterial OmpA protein, wherein (i) the native N-terminus and the native C-terminus of the bacterial OmpX protein or the bacterial OmpA protein are joined by a linker, wherein the linker is a short flexible peptide, and (ii) a non-native N-terminus and a non-native C-terminus are derived from an extracellular loop of the bacterial OmpX protein or the bacterial OmpA protein, wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly, and (b) a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus of the circularly permuted bacterial outer membrane protein, wherein the passenger polypeptide is displayed on the outer membrane of the host cell. - View Dependent Claims (11, 12, 13, 14, 15, 18, 21)
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Specification