Display library process
Disclosed are methods for identifying desired members from a display libraries, including bacteriophage display libraries. Display library members can be amplified in the presence of a target compound so that cycles of selection can be rapidly completed.
1. A method of selecting a binding protein that binds to a target, the method comprising:
(a) contacting a first plurality of diverse phage particles to a target to form phage-target complexes, wherein each phage particle of the first plurality displays a heterologous protein component on its surface and each phage includes a nucleic acid encoding the heterologous protein component, and wherein each phage-target complex comprises a phage from the first plurality, the heterologous protein component displayed on which binds the target;
(b) separating the phage-target complexes from unbound phage particles;
(c) contacting the separated phage-target complexes with a ligand under conditions wherein the ligand causes dissociation of one or more of the phage-target complexes to release free phage particles;
(d) isolating the free phage particles, wherein the heterologous protein components displayed on the surface of the free phage particles are binding proteins to the target; and
(e) infecting host cells with the isolated free phage particles from step (d) to produce a second plurality of phage particles,
wherein step (e) is performed in the presence of the target.
2. The method of claim 1 , wherein step (e) comprises: maintaining the host cells under conditions whereby the free phage particles are replicated, thereby producing the second plurality of phage particles.
3. The method of claim 1 , further comprising repeating steps (a)-(e) for one or more cycles.
4. The method of claim 1 , wherein the ligand binds to the target.
5. The method of claim 4 , wherein the target is a protein and the ligand binds to an epitope of the target.
6. The method of claim 5 , wherein the concentration of the ligand in step (c) is greater than its dissociation constant (Kd) for the target.
7. The method of claim 1 , wherein the ligand binds to the phage.
8. The method of claim 7 , wherein the target is a protein and the ligand is structurally homologous to at least a segment of the target.
9. The method of claim 1 , wherein step (e) further comprises removing the ligand from a culture media where the infected host cells are maintained.
10. The method of claim 1 , wherein the target is immobilized to a support.
11. The method of claim 10 , wherein the target is immobilized to the support via a non-covalent interaction.
12. The method of claim 10 , wherein the target is covalently immobilized to the support.
13. The method of claim 1 , further comprising recovering the second plurality of diverse phage produced in step (e).
14. The method of claim 1 , wherein during step (e), fewer than 5000 progeny phage are produced for each phage particle that infects one of the host cells.
15. The method of claim 1 , wherein step (e) is completed in less than 4 hours.
16. The method of claim 2 , wherein during step (e), the host cells divide less than seven times.
17. The method of claim 2 , wherein the step (e) involves a change in temperature.
18. The method of claim 1 , wherein each phage of the first plurality comprises genes sufficient for phage replication in a host cell.
19. The method of claim 1 , wherein each phage of the first plurality comprises a phagemid, and wherein step (e) is performed in the presence of helper phages.
20. The method of claim 1 , wherein the host cells are cells of a mutator strain.