Polypeptide display libraries and methods of making and using thereof
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.
1. A composition comprising:
a fusion protein, the fusion protein comprising
a bacterial outer membrane protein,
wherein the bacterial outer membrane protein is OmpA or OmpX,
wherein a native N-terminus and a native C-terminus of the bacterial outer membrane protein are joined by a linker, and
wherein the bacterial outer membrane protein comprises a non-native N-terminus and a non-native C-terminus derived from an extracellular loop of the bacterial outer membrane protein; and
a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus.
2. The composition of claim 1 , wherein the passenger polypeptide is a substrate for an enzyme.
3. The composition of claim 1 , wherein the passenger polypeptide comprises a peptide ligand.
4. The composition of claim 1 , wherein the fusion protein comprises a label.
5. The composition of claim 4 , wherein the label is a fluorescent agent.
6. The composition of claim 4 , wherein the label is C-terminal to the fusion protein.
7. The composition of claim 4 , wherein the label is N-terminal to the fusion protein.
8. The composition of claim 4 , wherein the label comprises an affinity tag.
9. The composition of claim 1 , wherein the passenger polypeptide is linked or fused directly to the non-native C-terminus of the bacterial outer membrane protein.
10. The composition of claim 1 , wherein the passenger polypeptide is linked or fused directly to the non-native N-terminus of the bacterial outer membrane protein.
11. The composition of claim 1 , wherein the fusion protein is disposed in an outer membrane of a host cell, and wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly.
12. The composition of claim 11 , wherein the non-native N-terminus is positioned extracellularly.
13. The composition of claim 11 , wherein the non-native C-terminus is positioned extracellularly.
14. The composition of claim 11 , wherein both the non-native C-terminus and the non-native N-terminus are positioned extracellularly.
15. The composition of claim 11 , wherein the host cell is a bacterial cell, a yeast cell or a mammalian cell.
16. The composition of claim 11 , wherein the host cell is a bacterial cell.
17. The composition of claim 16 , wherein the bacterial cell is an Escherichia coli cell.
18. A polypeptide display library comprising:
a plurality of host cells, each member of the plurality comprising
a fusion protein disposed in an outer membrane of a host cell, the fusion protein comprising
a bacterial outer membrane protein,
wherein the bacterial outer membrane protein is OmpA or OmpX,
wherein a native N-terminus and a native C-terminus of the bacterial outer membrane protein are joined by a linker, and
wherein the bacterial outer membrane protein comprises a non-native N-terminus and a non-native C-terminus derived from an extracellular loop of the bacterial outer membrane protein; and
a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus of the bacterial outer membrane protein, wherein the passenger polypeptide is displayed on the outer surface of the host cell and at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly.
19. The polypeptide display library of claim 18 , wherein the passenger polypeptide is a substrate for an enzyme.
20. The polypeptide display library of claim 18 , wherein the passenger polypeptide comprises a peptide ligand.
21. The polypeptide display library of claim 18 , wherein the fusion protein comprises a label.
22. The polypeptide display library of claim 21 , wherein the label is a fluorescent agent.
23. The polypeptide display library of claim 21 , wherein the label is C-terminal to the fusion protein.
24. The polypeptide display library of claim 21 , wherein the label is N-terminal to the fusion protein.
25. The polypeptide display library of claim 21 , wherein the label comprises an affinity tag.
26. The polypeptide display library of claim 18 , wherein the passenger polypeptide is linked or fused directly to the non-native C-terminus of the bacterial outer membrane protein.
27. The polypeptide display library of claim 18 , wherein the passenger polypeptide is linked or fused directly to the non-native N-terminus of the bacterial outer membrane protein.
28. The polypeptide display library of claim 18 , wherein the non-native N-terminus is positioned extracellularly.
29. The polypeptide display library of claim 18 , wherein the non-native C-terminus is positioned extracellularly.
30. The polypeptide display library of claim 18 , wherein the host cells are bacterial cells, yeast cells or mammalian cells.
31. The polypeptide display library of claim 18 , wherein the host cells are bacterial cells.
32. The polypeptide display library of claim 31 , wherein the bacterial cells are Escherichia coli cells.